IL297291A - Polymer excipients for biopharmaceutical formulations - Google Patents

Description

POLYMER EXCIPIENTS FOR BIOPHARMACEUTICAL FORMULATIONS RELATED APPLICATIONS This application claim spriority to U.S. Application Serial No. 63/011,928, filed April 17, 2020, and U.S. Application Serial No. 63/159,306, filed March 10, 2021, the disclosure whics h are incorporated herein by reference in their entireties.

GOVERNMENT RIGHTS This inventio nwas made wit hgovernment support under Grant DK119254 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.

FIELD OF THE INVENTION The present disclosure relates to amphiphilic polyacrylamide-based copolymer excipient sthat can be used to reduc eor prevent aggregation of biologic molecules, such as proteins and peptides, and lipid-based vehicles in aqueous formulations at hydrophobic interfaces, thereby increasing the thermal stabilit ofy the molecul esin the formulation. The present disclosure also relates to formulations of a monomeric insulin, and co-formulations of insulin and other proteins. The formulations containing the copolymer excipient exhibit increased stabilit asy compared to current rapid acting mealtim insue lin.

BACKGROUND Over 40 million patients liv ewit hdiabetes worldwide and rely on insulin replacement therapy through dail ysubcutaneous insulin injections or insulin infusion pumps These. patients are unable to produce sufficient insulin required to promote cellula r glucose uptake for basic metabol icfunction and therefore must deliver calculated insuli n doses to manage glycemic excursions. Unfortunatel they, pharmacokinetics of current insulin formulations do not mimic endogenous insulin secretion, which can reach peak concentrations in 30 minutes in a non-diabetic individua l.Even current "rapid-acting" insulin analogues, designed for mealtim boluses,e exhibit delayed onset of action of 20-30 minutes peak, action at 60-90 minutes and, a total duration of action of 3-4 hours. These kinetics are an outcom eof the mixed association stat esof the insulin molecules in formulation. Commercial insulin formulations typically contain a mixture of insuli n hexamers, dimers , and monomers. While monomers are rapidl yabsorbed into the bloodstream after injection, dimers and hexamers are absorbed more slowly on account of their size and must dissociate into monomers to become active (FIG. 1). Further, the extende dduration of insulin action can make controlling post-prandial glycemi c excursions difficult and increases the risk of hypoglycemia, as insulin may remain on board even afte rthe mealtim glucose loade passes.

An insulin formulation that is absorbed rapidly from the subcutaneous space to more closel mimy ic endogenous post-prandial insulin secretion is needed to better control mealtim bloode glucose. A monomeric insulin formulati onwoul denable both faster onset and shortened duration of action, thus reducing the risk of post-prandial hypoglycemia by eliminating the subcutaneous depot of insulin hexamers (FIG. 1A and FIG. IB). However, monomeric insulin is unstabl ine formulati onand rapidly aggregates into amyloid fibrils, which are both inactive and immunogenic. Presently, zinc and phenolic preservatives are commonly used as excipients in insulin formulations because their propensity to promote insulin hexamer formation enables them to act as stabilizing agents. It is critical to develop a new clas sof excipient sthat are capable of improving insuli nstabilit iny the monomeric state to enable a viable ultra-fas actingt insulin formulation.

Insuli naggregation typically is initiated at hydrophobic interfaces, such as the air- liquid interface, where monomers undergo partial unfolding upon adsorption and can nucleate amyloid fibril formation (FIG. IC). The monomeric stat ise most susceptib leto aggregation because hydrophobic moieties typically shielded in the dimeric and hexameric association states are responsible for aggregation. Current zinc-free methods for monomeric insuli nstabilizati onhave relied on shieldin ghydrophobic interactions by covalentl ory non-covalentl atty aching hydrophilic polymers such as polyethyle neglycol) (PEG) or trehalose glycol-polym ersdirectl toy insulin. While these methods have proven effective at stabilizing insulin in formulation, they lead to increased circulation time in vivo, which is undesirable for an ultra-fas actingt insulin formulation. Further, whil e poly(ethylene glycol) polymers have been traditionall usedy in drug delivery because of their water solubilit andy biocompatibility, recent concerns around immunogenicit yare beginning to limit their use.

An alternative approach to insuli nstabilizati onexploits the propensity of amphiphili cpolymers to occupy the interface, preventing insulin-interface interactions (FIG. IC). Pol oxamers are an exampl eof polymer surfactant thats have been used to improve the stabilit ofy commercial insulin formulations (INSUMAN® U400, Sanofi- Aventis). Yet ,these poloxamer excipient scomprise a limited chemical space, exhibit a propensity to form micro-structures such as micelles in solution, and are susceptible to transitioning into gels at high concentrations and, as such, a stable ultra-fast monomeric insulin formulati onis still evasive.

Insuli nhas been used to treat diabetes for almost 100 years, yet existing rapid- acting insulin formulations do not have fast enough pharmacokinetics to maintain tight glycemic contro lduring times of rapid glucose fluctuati onsuch as at mealtimes.

Dissociation of the insulin hexamer, the primary association stat ofe insulin in rapid-acting formulations, is the rate limiting step that leads to delayed onset and extended duration of action. A formulati onof insulin monomers woul dmore closely mimic endogenous post- prandial insulin secretion, but using known formulati onstrategies, monomeric insulin is unstabl ine soluti onand rapidly aggregates into amyloid fibrils.

Patients with certain types of diabetes lack sufficient pancreatic beta cell mass and/or function to produce both endogenous insulin and amylin. In non-diabeti c individual insulis, nand amylin work synergistically to contro lpost-prandial glucos e; amylin delays gastric emptying and suppresse glucagons action, while insulin promotes cellular glucose uptake. Studies have shown that dual-hormone replacement therapy wit h insulin and amylin results in improved glycemic outcomes for patients wit hdiabetes, including a 0.3% reduction in HbAlc compared to treatment wit hinsulin alone. However, treatment of type 1 diabetes over the last 100 years has primarily focused on insuli n replacement. While a commerciall yavailable amylin analog (pramlintide exists) ,only 1.5% of patients who woul dbenefit from amylin replacement therapy had adopted it by 2012. This is primarily due to formulati onchallenges that result in the need for a burdensom eseparate injection of amylin in additio ton insulin at mealtimes.

Amylin is highly unstab leand rapidly aggregates to form inactive and immunogenic amyloid fibrils. Pramlintide, an amylin analog, has three amino acid modifications to reduc eits propensity to aggregate into amyloid fibrils, thus improving its shelf-life, but is formulated at pH=4 making it incompatible to be mixed wit hinsuli n formulations (typicall pH~7).y Further, in typical clinical administrations insulin and pramlintide have disparat epharmacokinetics ,which is in contrast to endogenous co- secretion of the two hormones from the beta-cells following the same diurnal patterns. The difference in absorption kinetics when delivered exogenousl yresul tsfrom the different association states of insulin and pramlintide in formulati on(FIG. 15 A). Pramlintide only exists as a monomer, while insulin formulations contain a mixture of hexamers, dimers, and monomers. The mixture of insulin association stat esresults in delayed absorption and prolonged duration of insulin action.

SUMMARY Provided in the present disclosure are polyacrylamide-bas copolymered excipients that contain a water-soluble carrier monomer wit han acrylamide reactive moiety and a functional dopant monomer wit han acrylamide reactive moiety. The copolymers have been found to reduc eor prevent aggregation of biologic molecules and lipid-based vehicle s in aqueous formulations at hydrophobic interfaces. Thus, the biologic molecules and lipid - based vehicles in the formulations containing the polyacrylamide-bas edcopolymers exhibit increased stability, such as increased thermal stabilit y,as compared to the same formulations that do not contain the polyacrylamide-based copolymers. The polyacrylamide-bas edcopolymer scan be used wit hany biologic molecule or lipid-base d vehicle that is susceptible to aggregation in an aqueous medium, including but, not limited to, proteins ,such as antibodie sand fragment sthereof, cytokines ,chemokines, hormones, vaccine antigens, cancer antigens, adjuvants and, combinations thereof. In some embodiments, the protein is a monoclonal antibody. In some embodiments, the polyacrylamide-bas edcopolymers reduc eor prevent aggregation of a protein susceptibl e to aggregation in an aqueous medium The. polyacrylamide-based copolymers can also be used wit hlipid-based vehicles that are susceptib leto aggregation in an aqueou smedium , including but, not limited to, liposomes, lipid nanoparticle s,polymerosomes and, micelles , to prevent or reduc eaggregation.

In some embodiments, the protein is insulin. In some embodiments, the polyacrylamide-bas edcopolymer is used in the development of an ultra-fas absorbint g insulin lispro (UFAL) formulation, which remains stabl undere stresse daging conditions for 25 ± 1 hours, compared to 5 ± 2 hours for commercial fast-acting insulin lispro formulations (HUMALOG®). In a swine model of insulin-deficient diabetes, UFAL exhibited peak action at 9 ± 4 minutes while commercial HUMALOG® exhibited peak action at 25 ± 10 minute s.These ultra-fas kinetit cs make UFAL a promising candidate for improving glucose control and reducing burden for patients wit hdiabetes.

According to exemplary embodiments, the polyacrylamide-based copolymer excipient senable stabl formulatie onof an ultra-fas actingt monomeric insulin. In some embodiments, these excipient sare syntheti ccopolymers composed of a water-solubl e carrier monomer, chosen to aid polymer solubility, and a functional dopant monomer, which affords the ability to screen a wide chemical space unexplored in current surfactant excipients. The dopant monomer is hypothesized to promote polymer-interface interactions reducing, insulin-insulin interactions at the interface, and thus improving insulin stabilit Iny. some embodiments, precision high-throughpu synthest isusing reverse additional fragmentation transfer (RAFT) polymerizatio isn employed to generate a library of over 100 polyacrylamide-bas copolymed ers. In the present disclosure it ,is demonstrat ed that the polyacrylamide-based copolymers enable the stabl eformulati onof monomeric insulin lispro and that this ultra-fast absorbing insulin lispro (UFAL) formulati onexhibits pharmacokinetic sthat are 2-fold faster than commercial fast-acting insulin formulations in a swine model of insulin-deficient diabetes.

In addition, in some embodiments, the polyacrylamide-based copolymers enable the stable co-formulation of monomeric insulin lispro and pramlintide wit hsynchronized ultrafas insult in-pramlintide pharmacokinetics that resul int better glycemic contro lin a mealtim sime ulation. This co-formulation has potential to improve glucose management and reduc epatient burden in clinical applications using it whenever rapid acting insulin woul dotherwis bee used. The herein described copolymer excipient scan also be applied more broadly to improve the thermal stabilit ofy protein formulations incl, uding insuli n formulations, as simple "drop-in" excipients, without altering their bioactivit y, pharmacokinetic s or pharmacodynamics. For example, the copolymer excipients described herein can be used as a drop-in excipient in combination wit hother formulati on approaches that are intended to alter or modulate the pharmacokinetic sof the protein formulation.

Also provided in the present disclosure is a polyacrylamide-based copolymer comprising a water-soluble carrier monomer comprising an acrylamide reactive moiety; and a functional dopant monomer comprising an acrylamide reactive moiety; wherein the weight percent (wt%) of the water-solubl carre ier monomer is about 70% to about 98%; the weight percent (wt%) of the functional dopant monomer is about 2% to about 30%; the average molecula weighr t (Mn) of the polyacrylamide-based copolymer is about 1,000 g/mol to about 30,000 g/mol ;and the degree of polymerization is about 10 to about 250.

As disclose hereind , high throughput controll edradical polymerization techniques were implemented to generate a large library of polyacrylamide-bas edcopolymer excipients. Non-limiting example sof polyacrylamide-bas edcopolymers as provided herein include a polyacrylamide-base copolymerd comprising a water-soluble carrier monomer selected from the group consisting of 7V-(3-methoxypropoyl)acrylami de (MPAM), 4-acryloylmorpholine (MORPH), 7V,7V-dimethylacrylami de(DMA), N- hydroxyethyl acrylamide (HEAM), and acrylamide (AM); and a functional dopant monomer selected from the group consisting of A-[tris(hydroxymethyl )- methyl]acrylamide (TRI), 2-acrylamido-2-methylpropane sulfoni cacid (AMP), (3- acrylamidopropyl)trimethylammonium chloride (TMA), A-isopropylacrylami de(NIP), N- tert-but acrylylamide (TEA), and N-phenylacrylamide (PHE).

Provided in the present disclosure is a composition comprising about 0.005 wt% to about 0.2 wt% of a polyacrylamide-bas edcopolymer comprising about 70% to about 95% by weight of a MORPH carrier monomer; and about 5% to about 30% by weight of a NIP dopant monomer; and about 100 U/mL insulin, or an analog thereof.

Also provided in the present disclosure is a composition comprising about 0.005 wt% to about 0.2 wt% of a polyacrylamide-based copolymer comprising about 70% to about 95% by weight of a MORPH carrier monomer; and about 5% to about 30% by weight of a NIP dopant monomer; about 100 U/mL insulin, or an analog thereof; and about 0.01 mg/mL to about 0.1 mg/mL pramlintide.

Also provided is a method of treating diabetes in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition comprising a polyacrylamide-bas edcopolymer of the present disclosure.

Also provided is a method of managing the blood glucose level in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition comprising a polyacrylamide-bas edcopolymer of the present disclosure.

Also provided is a method for increasing stabilit ofy a formulati oncontaining a biologic molecule, comprising adding about 0.005 wt% to about 5 wt% of a polyacrylamide-bas edcopolymer of the present disclosure to the formulation.

Also provided is a method for increasing stabilit ofy a protein formulatio n, comprising adding about 0.005 wt% to about 5 wt% of a polyacrylamide-based copolymer of the present disclosure to the protein formulation.

Also provided is a method for increasing stabilit ofy a formulati oncontaining a lipid-based vehicle , comprising adding about 0.005 wt% to about 5 wt% of a polyacrylamide-bas edcopolymer of the present disclosure to the formulation.

Also provided is a method for reducing the rate of aggregation of a biologic molecule in an aqueous composition, comprising adding about 0.005 wt% to about 5 wt% of a polyacrylamide-based copolymer of the present disclosure to the formulation.

Also provided is a method for reducing the rate of aggregation of a protein in an aqueous composition, comprising adding about 0.005 wt% to about 5 wt% of a polyacrylamide-bas edcopolymer of the present disclosure to the protein formulation.

Also provided is a method for reducing the rate of aggregation of a lipid-base d vehicle in an aqueous composition, comprising adding about 0.005 wt% to about 5 wt% of a polyacrylamide-based copolymer of the present disclosure to the formulation.

DESCRIPTION OF THE DRAWINGS FIG. 1 illustrate a schemes of absorption kinetics of the various association state s of insulin. FIG. 1A: Commercial "rapid-acting" insulin formulations contain a mixture of insulin hexamers, dimers ,and monomers. Only the monomeric form of insulin is active, thus, the dissociation from the hexamer to the monomer is rate limiting for therapeutic action. An ultra-fas insult in formulati onwoul dcontain primarily insuli nmonomers and no insulin hexamers for rapid insuli nabsorption after subcutaneous administration. FIG.

IB illustrat thees mixture of insulin hexamers, dimers, and monomers in commercial rapid- acting insulin formulations that resul tin extended duration of insuli naction when delivered subcutaneously Insuli. nmonomers are absorbed in approximately 5-10 minutes, dimers are absorbed in 20-30 minutes, and hexamers can take 1-2 hours to be absorbed and result in prolonged insulin action accordingly. A primarily monomeric insuli n formulati onwoul reduced time to onset and resul int shorter duration of insulin action for better management of blood glucos ate mealtimes. FIG. IC illustrate a hexams er-free ultra- fast insulin formulati onthat will face stabilit chally enges due to the propensit yfor insuli n monomers to aggregate into amyloid fibrils. At the air-water interface (shown on the left ), unfolding of insulin molecules and exposure of hydrophobic domains during insulin- insulin interaction promotes amyloid fiber formation. Stabilizing polymer excipients are drawn to the air-liquid interface (shown on the right), impeding the unfolding of insuli n molecules and the interfacial nucleation of insulin amyloidosis.

FIG. 2 illustrate a sscheme of polymer excipient library design. A library of statistica acrlylamide copolymers wit ha target degree of polymerizatio (DP)n of 50 were synthesized through controll edcopolymerization using RAFT. Copolymer combinations consist of one carrier monomer: acryloylmorpholi ne (MORPH), methoxypropylacrylami de (MPAM), dimethylacrylam ide (DMA), hydroxy ethylacrylamide (HEAM), or acrylamide (AM). Each copolymer also contains one dopant monomer: tris(hydroxymethyl)-methylacryla mide (TRI), acrylamidomethylpropane sulfoni cacid (AMP), acrylamidopropyltrimethylammoni um chloride (TMA), n-isopropylacrylami de(NIP), tertbutyl acrylamide (TEA), or phenyl acrylamide (PHE). Each carrier-dopant combination was repeated at low, medium and high dopant loadings: NIP at 6.7, 13.3, and 20 wt.%, TRI at 5, 10, and 15 wt.%, and AMP, TMA, TEA, PHE at 3.3, 6.7, and 10 wt.%.

FIG. 3 illustrate thes 1H NMR spectroscopy and SEC traces to valida teSEC wt.% measurement. 1HNMR spectroscopy of synthesized copolymers with MORPH as a carrier and PHE as a dopant is shown. MORPH-PHE6.7% received inadequate MORPH additio n on account of the high viscosity of the monomer, yielding a higher than expected loading of PHE in the final copolymer. The experimental loading was confirmed by SEC traces provided in the inset figure, where MORPH-PHE6.7% was determined to be a lower molecula weir ght than MORPH-PHE33% and MORPH-PHE10%. This decrease in molecula weir ght is used to determine experimental loading sof MORPH and PHE in the final copolymers DRI. refers to differential refractive index measured using SEC.

FIG. 4 illustrat SECes traces of polymers from the initial copolymer library synthesis .From left to right, the carrier monomers are: acrylamide (AM), N,N- dimethylacrylamide (DMA), N-hydroxyethyl acrylamide (HEAM), 4-acryloylmorpholine (MORPH), and A-(3-methoxypropyl)acrylam ide(MPAM). AM-based copolymers were measured on an aqueou sSEC, whereas the others were measured on a DMF SEC. The top row depicts overlays of all synthesized copolymers .Each subsequent row shows the functional dopant loadings for the copolymers. The three copolymerizations for each carrier-dopant pair are low loading, medium loading, and high loading. The functional dopants (abbreviation; low, medium , and high loading) are as follows: N- [tris(hydroxymethyl)methyl]acryla mide(TRI; 5, 10, 15 wt.%), (3- acrylamidopropyl)trimethylammonium chloride solution (TMA; 3.3, 6.7, 10 wt.%), 2- acrylamido-2-methylpropane sulfonic acid (AMP; 3.3, 6.7, 10 wt.%), N- isopropyl acrylamide (NIP; 6.7, 13.3, 20 wt.%), N-/c/7-butylacrylami (TEA;de 3.3, 6.7, 10 wt.%), and N-phenyl acrylamide (PHE; 3.3, 6.7, 10 wt.%).

FIGS. 5A-5G illustrat a erecombinant insuli nstabilit screy en wit ha polymer excipient library. Time to aggregation of recombinant insulin (100 U/mL) formulated wit h carrier homopolymers (0.1 wt.% )or a Pluroni cL-61 control (0.1 wt.%), which has similar composition to Pol oxamer 171 used in commercial insulin formulations is shown in FIG. 5A. Time to aggregation of recombinant insulin (100 U/mL) formulat edwit hcarrier- dopant polymers (0.1 wt.% )wit hDMA (FIG. 5B), AM (FIG. 5C), HEAM (FIG. 5D), MP AM (FIG. 5E), and MORPH (FIG. 5F) carriers is also shown. Dopants and target weight percentages are listed on the x-axis. FIG. 5G is a heat-map of the top performing excipient for each carrier-dopant combination (the longes ttime to aggregation). Dotted black square sindicate the top dopant-carrier combinations, which were selected for furthe r screening. These assays assess the aggregation of proteins in formulati onover time during stresse agingd (continuou agits ation at 37 °C by monitoring changes in absorbance at 540 nm. Data shown are average time to aggregation (n=3; mean ± s.d.) where aggregation is defined as a 10% increase in absorbance.

FIG. 6 illustrate aqueous sSEC elution profiles for commercial HUMALOG® and UFAL formulations. These traces illustrate the primarily monomeric insulin association state of UFAL by the longer elution time correlating wit hlower effective molecular weight.

FIGs. 7A-7D illustrate SEC traces of copolymers from the second screen targeting DP 50. FIG. 7A shows MP AM copolymerized wit hPHE. FIG. 7B shows MPAM copolymerized wit hNIP. FIG. 7C shows MORPH copolymerized with PHE. FIG. 7D shows MORPH copolymerized wit hNIP.

FIGs. 8A-8D illustrat a estabilize dultra-fas absorbint g insuli nlispro (UFAL) formulati onusing polyacrylamide-bas edcopolymer excipients. FIG. 8A shows insulin association states in HUMALOG® (top) and UFAL (bottom) as determined by MALS.

FIG. 8B is a UFAL stabilit screey n wit ha polymer excipient library. Time to aggregation of UFAL (100 U/mL) formulated wit hpolymer excipients from the second screen (0.01 wt.% )is shown. Polyacrylamide-based copolymers excipients comprised of MPAM and MORPH carrier polymers wit hvaried weight percent of dopants PHE (top) or NIP (bottom). FIG. 8C depicts representative absorbance traces showing UFAL stabilit wheny formulat edwit hMORPH-NIP23% compared to controls of UFAL wit hno polymer excipient, and HUMALOG®. These assays assess the aggregation of proteins in formulati onover time during stressed aging (i.e., continuous agitation at 37°C) by monitoring changes in transmittance at 540 nm. Data shown are average time to aggregation (n=3; mean ± s.d.) where aggregation is defined as a 10% increase in absorbance. FIG. 8D shows diffusion-ordered NMR Spectroscopy (DOSY) of UFAL wit h polymer excipient MORPH-NIP23%. DOSY provides insight into the insuli nassociation state and the insulin and polymer rates of diffusion in formulation. Diffusion characteristi csdemonstrat thate lispro and MORPH-NIP23% diffuse at different rates and are not associated, suggesting that disruption of interfacia linteractions are the primary contributor to the observed stabilizing effects.

FIGs. 9A-9B illustrate in vitro and in vivo formulati onbioactivity of HUMALOG®, UFAL, aged HUMALOG® (12 h shaking at 37 °C), and aged UFAL (12 h shaking at 37 °C). (FIG. 9A) In vitro activity was tested by assaying for phosphorylation of Ser473 on AKT. Data shown are mean ± s.e.m .for n=3 experimental replicates. Resul tswere plotted as a ratio of [pAKT]/[AKT] for each sampl e(n=3 cellular replicates and) an EC50 regression [log(agonist) vs. response (three parameters)] was plotted using GraphPad Prism 8. (FIG. 9B) In vivo bioactivity was assessed in diabetic mal eSprague Dawley rats.

Rats fasted for 4 to 6 h received a subcutaneous injection of insulin (1.5 U/kg) and glucos e measurements were taken using a handheld glucos monie tor every 30 minutes for 4 hours. 16 rats were randomly assigned to two groups :(i) HUMALOG® (n=8) and (ii) UFAL (n=8). Within each group each rat received both the fresh and aged formulations on separate days. The order the formulations were given was randomized. It is hypothesized that the loss of activit fory aged HUMALOG® observed in the blood glucose assay, but not the AKT assay, is a result of reversible insuli naggregation. When this aged formulati onundergoes significant dilution for the in vitro AKT assay, these aggregates dissociate into active insulin, whereas the minimal diluti onnecessary for accurate dosing in rats is not enough for this dissociation to occur and the insulin aggregates resul int the observed los sof activity. Data shown are mean ± s.e.m.

FIGs. 10A-10M illustrat pharmacokie netics and pharmacodynamics of monomeric insulin in diabetic swine. Diabetic femal epigs received subcutaneous administration of therapies comprising either (i) commercial HUMALOG® or (ii) UFAL formulat edwit h polymer. Pigs were dosed with insulin according to their individual insuli nsensitiviti toes decrease their glucose levels by approximatel y200 mg/dL. FIG. 10A: Blood glucose measurements in pigs after insulin dosed subcutaneously. FIG. 10B: Pharmacokinetics of insulin lispro in mU/L following s.c. injection. FIG. 10C: Total exposure represented by area under the curve for 210 minutes. FIGs. 10D-10I: Percent exposure at variou stim e points (AUC1/AUC210). FIG. 10J: Pharmacokinetics for each pig were individually normalized to peak concentrations and normalized values were averaged for lispro concentration for each treatment group. FIG. 10K: Time to reach 50% of peak lispro concentration (onset). FIG. 10L: Time to reach peak lispro concentration. FIG. 10M: Time for lispro depletion to 50% of peak concentration. FIGs. 10A, 10B, and 10J: Error bars indicate mean ± s.d. with n=5 for all groups .FIGs. 10D-10I: Error bars indicate mean ± s.e.m .wit hn=5 for all groups. Bonferroni post-hoc tests were performed to account for comparisons of multiple individual exposure timepoints and significance and alpha was adjust ed(alpha = 0.008). (FIGs. 10C, 10K-10M) Error bars indicate mean ± s.e.m. wit h n=5 for all groups (alpha=0.05). Statistical significance was determined by restricted maximum likelihood (REML) repeated measures mixed model.

FIGs. 11A-11B illustrat bloode glucos eof monomeric insulin in diabetic pigs.

Diabetic femal epigs received subcutaneous administrati onof therapies comprising either (i) commercial HUMALOG® or (ii) UFAL formulated wit hpolymer. FIG. 11 A: Scheme of subcutaneous injection sit ebehind the foreleg of the pig. Pigs have tight skin and subcutaneous tissue that is very similar to humans ,making them the most relevant preclinical model for studying pharmacokinetic sof biopharmaceuticals following subcutaneous administration. Pigs are sufficiently large for insulin to be administered accurately using standard concentrations (100 U/mL), ensuring the observed pharmacokinetic sare not skewed by dilution effects. FIG. 11B: Pigs were dosed wit h insulin according to their individual insuli nsensitiviti toes decrease their blood glucose concentrations by about 200 mg/dL. Blood glucos emeasurements in pigs after insulin dosed subcutaneously Error. bars indicate mean ± s.d. wit hn=5 for all groups.

FIGs. 12A-12E illustrate pharmacokineti cmodelling of UFAL in humans .FIG. 12A: A model of insulin plasma concentrations after injection in human patients was adapted from Wong et al. (J. Diabetes Set. TechnoL (2008) 2:658-671). Rapid-acting insulin analogues are injected into the subcutaneous space (7mj), then dissociate and diffuse into the interstitium (G) where they are then absorbed into the plasm a^2) and ultimatel y cleared (U). FIG. 12B: Normalized pharmacokinetic data for HUMALOG® and UFAL in diabetic pigs modeled using a least squares fit to determine k\, ki, and fa in pigs (FIG. 14 and Table 6). FIG. 12C: Human clinical HUMALOG® pharmacokinetic data compared to modeled rapid-acting insulin (RAI) analogue kinetics (using known human parameters, Table 6), and the predicted kinetics of UFAL in humans .UFAL human pharmacokinetics were predicted by first fitting the pig pharmacokinetic data for HUMALOG® and UFAL.

The human UFAL pharmacokinetics was then plotted by using the estimat edk\ wit hthe known ki and fa parameters. FIG. 12D: Model predicted kinetics of RAI and UFAL compared to HUMALOG® kinetics in publishe dclinical studies, time to 50% peak up (left), time to peak (middle) and duration of action time to 50% of peak down (right). FIG. 12E: Comparison of the model predicted time to peak for UFAL in humans compared to human clinical data for commercial rapid-acting insulin formulations (see, for example: Heise et al., Diabetes Obes. Metab. (2017) 19:208-215; Andersen et al., Diabetes Obes.

Metab. (2018) 20:2627-2632; Rave et al., Diabetes Care (2006) 29:1812-1817).

FIG. 13 illustrat AUMCes /AUC for UFAL and HUMALOG® in diabetic pigs.

Diabetic femal epigs received subcutaneous administrati onof therapies comprising either (i) commercial HUMALOG® or (ii) UFAL formulat edwit hpolymer. Pigs were dosed wit h insulin according to their individual insuli nsensitiviti toes decrease their blood glucose concentrations by about 200 mg/dL. For subcutaneous administration, area under the moment curve (AUMC) divide dby area under the curve (AUC) is equal to the mean residence time (MRT) + mean absorption time (MAT). AUMC/AUC = MRT + MAT.

Error bars indicate mean ± s.e.m. wit hn=5 for all groups (alpha=0.05). Statistical significance was determined by restricted maximum likelihood (REML) repeated measures mixed model.

FIGs. 14A-14C illustra pharmate cokinetic outputs from model fitting compared to experimental pharmacokineti cdata for HUMALOG® and UFAL in diabetic swine .FIG. 14A: Time to 50% of peak up. FIG. 14B: Time to the peak. FIG. 14C: Time to 50% of the peak down. Stars indicate predicted pharmacokinetic timepoints from the model. Data points are the same used in FIGs. 10K-10M.

FIGs. 15A-15F illustrat thee formulati onkinetics and stability. FIG. 15A: Current dual-hormone replacement of insulin and pramlintide requires two separate injections at mealtimes. Not only is this additiona linjection burdensom e,but there is a kinetic mismatch between insulin and pramlintide when delivered exogenousl ycompared to endogenous co-secretion from the beta-cell s.This results from the mixed insuli n association stat espresent in rapid-acting insulin formulations where monomers and dimers are rapidly absorbed, but the slow dissociation of the insulin hexamer causes extended duration of action. FIG. 15B: A single injection co-formulati onof monomeric insulin and pramlintide woul dreduce patient burden, and have better pharmacokineti coverlap that more closely mimics endogenous secretion from the healthy pancreas. FIG. 15C: Amphiphili cacrylamide copolymer excipients can be used to stabilize an insulin- pramlintide co-formulation. These excipients preferentially adsorb onto the air-water interface, displacing insulin and/or pramlintide and preventing the nucleation of aggregation events that initiat eamyloid fibril formation. FIG. 15D: Co-formulation components. FIG. 15E: Insuli nassociation stat esin HUMALOG® (top) ;adapte dfrom 57, compared to zinc-free lispro with phenoxyethanol (0.85 wt.%) and glycerol (2.6 wt.% ) (bottom). FIG. 15F: Formulation stabilit iny a stressed aging assay (continuous agitation, 37 °C) of (i) HUMALOG®, (ii) HUMALOG® + pramlintide (1:6 pramlintide:lispro), (iii) zinc-free lispro (100 U/mL lispro, 0.85 wt.% phenoxyethanol, 2.6 wt.% glycerol, 0.1 mg/mL M0Ni23%), (iv) Co-formulation (100 U/mL lispro, 1:6 pramlintide:lispr 0.85o, wt.% phenoxyethanol 2.6, wt.% glycerol, 0.1 mg/mL M0Ni23%). Change in transmittance is shown from baseline transmittanc Aggregate. ion is defined as a change in transmittance >10%.

FIG. 16 illustrate SEC-MAs LS elution profiles of the distribut ionof insulin lispro aggregation state swit h number-averaged molecula rweight and weight-averaged molecula weir ght used to calculat percentagee of insulin association states.

FIG. 17 illustrat initesial transmittanc fore HUMALOG® + pramlintid e,and co- formulati on(100 U/mL lispro, 1:6 pramlintide:lisp ro,0.85 wt.% phenoxyethanol, 2.6 wt.% glycerol 0.1, mg/mL M0Ni23%). Initial transmittance values for (i) HUMALOG® + pramlintide contro lgroup and (ii) co-formulati onin the stabilit assy ay (FIG. 15F) are shown as the change in transmittance of (Average initial HUMALOG® transmittance) - formulati oninitial transmittanc e.The decreased transmittanc eobserved for the HUMALOG® + Pramlintid sampe les before the aging study indicates that there is poor solubilit wheny these two formulations are mixed. In comparison, the initial transmittance for the co-formulation is optically clear to the eye and shows litt ledifference from initial HUMALOG® transmittance.

FIGs. 18A-18D illustrat thee pharmacokinetic sand pharmacodynamics in diabetic rats. Fasted mal e diabetic rats (n=ll) received subcutaneous administrati onof (i) HUMALOG®, (ii) separate injections of HUMALOG® and pramlintide, or (iii) insulin- pramlintide co-formulation. Insuli nadministration was immediatel followey witd horal gavage with a glucos esolution (1 g/kg). Each rat received all treatment groups. Change in blood glucose levels from baseline following treatment is shown in FIG. 18A. FIGs. 18B- 18C show the pharmacokinetics of insulin lispro (FIG. 18B) or pramlintide (FIG. 18C).

FIGs. 19A-19M illustrate onset and duration of action in diabetic rats .Faste dmale diabetic rats (n=ll )received subcutaneous administration of (i) HUMALOG®, (ii) separate injections of HUMALOG® and pramlintid e,or (iii) insulin-pramlintide co- formulation. Insuli nadministrati onwas immediatel folly owed wit horal gavage wit ha glucose soluti on(1 g/kg). Each rat received all treatment groups .Pharmacokinetics for each rat was individual normalily zed to the peak serum levels and the normalized values were averaged for insulin lispro (FIG. 19A) or pramlintide (FIG. 19J). Exposure onset defined as time to 50% of the peak up for insulin lispro (FIG. 19B) or pramlintide (FIG. 19K). Exposure peak for insulin lispro (FIG. 19C) or pramlintide (FIG. 19L). Exposure onset defined as time to 50% of the peak up for insuli nlispro (FIG. 19D) or pramlintide (FIG. 19M). Fraction of lispro exposure as a ratio of AUC1/AUC120 at FIG. 19E, t=6; FIG. 19F, t=15; FIG. 19G, t=30; FIG. 19H, t=45; FIG. 191, t=60. Statistica signil ficance was determined by restricted maximum likelihood repeated measures mixed model. Tukey HSD post-hoc test weres applied to account for multiple comparisons (FIGs. 19b-191,19K, 19M). Bonferroni post hoc test weres performed to account for comparisons of multiple individual exposure time points, and significance and a were adjust ed(a= 0.01) (FIGs. 19E-19I).

FIGs. 20A-20C illustrate lispro area under the curve and exposure ratios. Total lispro exposure as area under the pharmacokinetic curve (FIG. 20A). Fraction of lispro exposure as a ratio of AUCt/AUC120 at FIG. 20B, t=3; FIG. 20C, t=9. Statistical significance was determined by restricted maximum likelihood repeated measures mixed model. Tukey HSD post-hoc test weres applied to account for multiple comparisons (FIGs. 20B-20C).

FIGs. 21A-21G illustrat pramle intide area under the curve and exposure ratios .

Total pramlintide exposure as area under the pharmacokineti ccurve (FIG. 21 A). Fraction of pramlintide exposure as a ratio of AUC1/AUC120 at FIG. 20B, t=6; FIG. 20C, t=9; FIG. 20D, t=15; FIG. 20E, t=30; FIG. 20F, t=45; FIG. 20G, t=60. Statistical significance was determined by restricted maximum likelihood repeated measures mixed model.

FIGs. 22A-22C illustrat thee pharmacokinetic overlap of formulations. Average normalized serum concentrations (for each rat, n=ll/group) for insulin and pramlintide when delivered as two separate injections and when delivered togethe asr a co-formulation are shown in FIGs. 22A and 22B, respectively. Overlap between the two curve swas defined as the total time spent above 0.5 for both insulin and pramlintide curves (width at half-peak height), shown as a ratio of the overlap time to the total width of both peaks (overlap ؛ (lispro + pramlintide - overlap) (FIG. 22C). Statistica sigl nificance was determined by restricted maximum likelihood repeated measures mixed model.

FIGs. 23A-23C illustrat gastrie cemptying in diabetic rats. Fasted mal ediabetic rats received subcutaneous administrati onof (i) HUMALOG®, (ii) separate injections of HUMALOG® and pramlintide, or (iii) insulin-pramlintide co-formulation. FIG. 23 A depicts a gastri c emptying experiment where insulin administration (2 U/kg) was immediatel folly owed wit horal gavage wit han acetaminophen slurr y(100 mg/kg). Each rat (n=l 1) received all treatment groups. Acetaminophen serum concentration is shown in FIG.23B. Time to peak exposure of acetaminophen serum concentration is shown in FIG. 23C. All data is shown as mean ± SE. Statistica signil ficance was determined by restricted maximum likelihood repeated measures mixed model. Tukey HSD post-hoc test wers e applied to account for multiple comparisons.

FIGs. 24A-24D illustrate mealtim sime ulations with glucose. Fasted mal ediabetic rats received subcutaneous administrati onof (i) HUMALOG®, (ii) separate injections of HUMALOG® and pramlintide, or (iii) insulin-pramlintide co-formulation. FIG. 24A depicts oral glucose challenge where insulin administrati on(0.75 U/kg) was immediate ly followed with oral gavage wit ha glucose soluti on(2 g/kg). Each rat (n=10) received all treatment groups .Change in blood glucose after administration is shown in FIG. 24B. Max change in glucos abovee baseline is shown in FIG. 24C. Max change in glucose below the baseline is shown in FIG. 24D. All data is shown as mean ± SE. Statistica signifil cance was determined by restricted maximum likelihood repeated measures mixed model Tukey.

HSD post-hoc test weres applied to account for multiple comparisons.

FIGs. 25A-25C illustrat inte erspecies pharmacokinetic sfor HUMALOG® and HUMULIN@. Normalized pharmacokinetic sfor commercial HUMALOG® and regular human insulin (ex. HUMULIN R) delivered in rats and humans is shown in FIG. 25A.

Time to peak exposure for each formulati onis shown in FIG. 25B and duration of action for each formulati onis shown in FIG. 25C. Differences in time to onset and time to peak in rats between rapid-acting and regular insulin formulations are minimal and difficult to detect How. ever, in humans ,these small differences translat toe distinc diffet rences in time to onset and time to peak. This suggests that the trend for more rapid action we observe for insulin in the co-formulations of the present application compared to HUMALOG® coul dtranslat inte o substanti diffeal rences in humans. Duration of action is defined here as peak width at 25% peak height (time 25% down - time 25% up). Rat data for HUMALOG® is taken from this study, and rat data for regular human insulin is adapted from previous work. Human HUMALOG® data is from three externa lstudi esand has been adapted from presentation in previous work. Human regular human insuli ndata is from three externa lstudi es(Pettis et al., Diabetes Technol. Ther. (2011) 13:443-450; Andersen et al., Diabetes Obes. Metab. (2018) 20:2627-2632; Plank et al., Diabetes Care (2002) :2053-2057; Linnebjerg et al., Clin. Pharmacokinet. (2020) 59:1589-1599).

FIGs. 26A-26C illustrat inte erspecies pharmacokinetic sfor HUMALOG® and monomeric lispro formulations. FIG. 26A shows normalized pharmacokinetic sfor commercial HUMALOG® delivered in rats, pigs and humans (left ),time to peak (middle), and duration of action (right). FIG. 26B shows normalized pharmacokinetics for monomeric insulin delivered in rats ,pigs and humans (left ),time to peak (middle), and duration of action (right). HUMALOG® shows increased time to onset and longer duration of action as you shift to species wit hmore complex subcutaneous architecture (rats < pigs < humans). In contrast, monomeric lispro has very simila ronset and duration of action betwee nrats and pigs. Since the difference between rats and pigs is minimal for this formulation, it is also likel ythat the difference between pigs and humans will be smal l.

Further, we observe that pramlintide - which only exists as a monomer - has very similar kinetics between pigs and humans. FIG. 26C shows normalized pharmacokinetic sfor commercial formulations in humans (left), time to peak exposure for each formulati on (middle), and duration of action for each formulati on(right). Even with the shift towards faster time to peak wit hnext generation rapid-acting insulins like Fiasp and Lyumjev there have not been similar increases in reducing duration of action. Duration of action is defined here as peak width at 25% peak height (time 25% down - time 25% up). Rat data is taken from the study described in Example 3 (monomeric lispro in rats was delivered as part of the co-formulation) Pig. data was adapted from previous work. Human HUMALOG® data is from four externa lstudie (Pets tis et al., Diabetes Technol. Ther. (2011) 13:443-450; Andersen et al., Diabetes Obes. Metab. (2018) 20:2627-2632; Plank et al., Diabetes Care (2002) 25:2053-2057; Linnebjerg et al., Clin. Pharmacokinet. (2020) 59:1589-1599).

Predicte dmonomeric lispro in humans has been adapted from pharmacokinetic modeling from previous work in pigs. For FIG. 26C, data is adapted from clinical studie ins humans for (i) regular human insulin (Rave et al., Diabetes Care (2006) 29:1812-1817; Lindholm et al., Diabetes Care (1999) 22:801-805; Heinemann et al., Diabetic Medicine (1996) 13:625-629), (ii) NOVOLOG® (Novo Nordisk) (Fath et al., Pediatr. Diabetes (2017) 18:903-910; Heise et al., Clin. Pharmacokinet. (2017) 56:551-559), (iii) Fiasp (Novo Nordisk) (Fath et al., Pediatr. Diabetes (2017) 18:903-910; Heise et al., Clin.

Pharmacokinet. (2017) 56:551-559), (iv) HUMALOG® (Eli Lily) (Pettis et al., Diabetes TechnoL Ther. (2011) 13:443-450; Andersen et al., Diabetes Obes. Metab. (2018) :2627-2632; Plank et al., Diabetes Care (2002) 25:2053-2057; Linnebjerg et al., Clin.

Pharmacokinet. (2020) 59:1589-1599), and (v) Lyumjev (Eli Lily) (Linnebjerg et al., Clin.

Pharmacokinet. (2020) 59:1589-1599; Shiramoto et al., J. Diabetes Invest. (2020) 11:672- 680). Predicted monomeric lispro in humans has been adapted from pharmacokinetic modeling from previous work in pigs (Mann et al., Sci. Transl. Med. (2020) 12:eaba6676).

FIGs. 27A-27C illustrat humane pharmacokinetic sfor various insulin formulations and shows normalized pharmacokinetics for pramlintide delivered in rats, pigs and humans (FIG. 27A), time to peak (FIG. 27B), and duration of action (FIG. 27C). The conservation of the ultra-rapid absorbance kinetics from rats to pigs and the simila pramlr intide kinetics betwee npigs and humans corroborates the model predicted kinetics for monomeric lispro in humans .Duration of action is defined here as peak width at 25% peak height (time 25% down - time 25% up). Rat data is taken from the study described in Exampl e3. Pig data was adapted from previous work (Maikawa et al., Nat. Biomed. Eng. (2020) 4:507-517).

Human pramlintide data is adapted from two external studi es(Kolterma net al., Diabetologia (1996) 39:492-499); Riddl eet al., Diabetes Obes. Metab. (2015) 17:904- 907).

FIGs. 28A-28D illustrat a escheme of cold chain and insulin aggregation mechanism . To maintain integrity, commercial insulin formulations must currently be transported and stored in refrigerated containers for the weeks-long duration of worldwi de distribut ion(FIG. 28A). FIG. 28B shows the aggregation mechanism of commercial insulin formulations. The insulin hexamer is at equilibrium with monomers in formulation.

These monomers interact at the interface, where the exposure of hydrophobic domains during insulin-insulin interaction nucleate amyloid fiber formation. FIG. 28C depicts the chemical structure of an example polyacrylamide-base dcopolymer excipient , poly(acryloylmorpholine77%-co-N-isopropylacrylamide2 (M0Ni23%).3%) Polyacrylamide- based copolymer excipients are amphiphilic copolymers that adsorb to interfaces ,reducing insulin-insulin interactions and delaying the nucleation of insulin amyloidosi (FIG.s 28D).

FIGs. 29A-29C illustr ateexperimental insight into the mechanism of polyacrylamide-bas edcopolymer excipient stabilization. FIG. 29A is an illustration of a proposed stabilizati onmechanism .In commercial HUMULIN®, monomers at the interface have associative interactions (top). Alone, M0Ni23% occupies the interface without the presence of insulin (middle ).In combination wit hHUMULIN® formulations M0Ni23%, disrupts insulin-insulin surface interactions, providing a mechanism for inhibiting aggregation (bottom). FIG. 29B shows surface tension measurements of HUMULIN®, M0Ni23% (0.01 wt.%) formulat edwit h formulati onexcipients, and HUMULIN® formulat edwith M0Ni23% (0.01 wt.% )(n=2). FIG. 29C shows interfacia lrheology measurements of HUMULIN®. Measurements for HUMULIN® formulat edwit hM0Ni23% (0.01 wt.%) fell below the resolution of the instrument, indicating that there is no protein aggregation at the interface (n=3).

FIG. 30 illustrat surfacees tension of 1 mg/mL (0.1 wt.% )polymer formulations.

Surface tension measurements of HUMULIN® (95 U), M0Ni23% (0.1 wt.% )formulat ed wit hglycerol (1.6 wt.% )and metacresol (0.25 wt.%), and HUMULIN® (95 U) formulat ed wit hM0Ni23% (0.1 wt.% )are shown.

FIGs. 31A-31H illustra thatte formulati onwit hpolyacrylamide-bas edcopolymers stabilizes insulin. FIG. 31 A: 1 mL of Commercial HUMULIN® or HUMULIN® wit hthe addition of polyacrylamide-bas edcopolymer excipient s(i) M0Phe6%, (ii) MpPhe8%, (iii) M0Ni23%were aliquote intod 2 mL glas svial sand aged at 37°C wit hconstant agitation (150 rpm) for 0, 2, 4, and 6 months. Addition al2 week and 1 month timepoints were added for the HUMULIN® control. All formulations were at a concentration of 95 U/mL (diluted so that copolymers coul dbe added to commercial HUMULIN®). FIG. 3 IB shows a transmittanc asse ay used to assess the aggregation of proteins in formulation over time by monitoring changes in transmittance at 540 nm (n=l per formulation timepoint). FIG. 3 IC shows in vitro activity by assaying for phosphorylation of Ser473 on AKT afte rstimulati on wit heither HUMULIN® or M0Ni23% at 0 month and 6 month timepoints. Insulin concentrations are shown as Log(ng/mL). FIG. 3 ID shows the Log(EC50) values for each formulation. Statistical significance was assessed using the Extra sum-of-squares F-test to determine if Log(EC50) differed between datasets. Data sets were compared in pairs, and Bonferroni post-hoc test weres used to adjus tfor multiple comparisons (alpha=0.008).

FIGs. 31E-31H depict circular dichroism spectra from 200-260 nm for each formulati on (diluted to 0.2 mg/mL in PBS) at each time point .For FIG. 3 IC, results shown are mean ± s.e plotte asd a ratio of [pAKT]/[AKT] for each sampl e(n=3 cellular replicates) and an EC50 regression (log(agonist) vs. response (three parameters)) was plotte usingd GraphPad Prism 8. For FIG. 3 ID, statistica signil ficance was assessed using the Extra sum-of- squares F-test to determine if Log(EC50) differed betwee ndatasets. Data sets were compared in pairs, and Bonferroni post-hoc test swere used to adjus tfor multiple comparisons (alpha=0.008).

FIGs. 32A-32B illustrate transmittance assays for 5 mg/mL and 1 mg/mL formulations 1 .mL of HUMULIN® wit hthe addition of polyacrylamide-based copolymer excipient s(i) M0Phe6%, (ii) MpPhe8%, (iii) M0Ni23% were aliquote intod 2 mL glas s vial sand aged at 37°C wit hconstant agitation (150 rpm) for 0, 2, 4, and 6 months. FIG. 32A shows 5 mg/mL polymer excipient in formulati on(0.5 wt.%) and FIG. 32B shows 1 mg/mL polymer excipient in formulati on(0.1 wt/%).

FIGs. 33 A-33F illustrat insule in activity after aging in diabetic rats. Fasted diabetic mal erats received subcutaneous administrati on(1.5 U/kg) of each insuli nformulati on HUMULIN® (FIG. 33A), HUMULIN® with M0Phe6% (FIG. 33B), HUMULIN® wit h MpPhe8% (FIG. 33C), or HUMULIN® wit hM0Ni23%(FIG. 33D) at each aging timepoint (0, 2, 4, 6 months). FIG. 33E is a comparison of each formulati onat t=0 months. In these assays, 32 rats were randomly assigned to one of the four formulati ongroups (n=8) and each rat received one dose of the formulati onat each aging timepoint in a random order.

Blood glucose levels were measured every 30 minutes using a handhel dglucose monitor and the change in blood glucos erelative to baseline glucose measurements were plotted.

The maximum difference in glucose from baseline (A glucose) was also plotte ford each formulati onas a measure of formulati onpotency. FIG. 33F shows the pharmacokinetics of HUMULIN® and HUMULIN® wit hM0Ni23% at t=0 and t=6 months. All data is shown as mean ± s.e. Statistical significance between max A glucose was assessed using a REML repeated measures mixed model wit hrat as a random effect and the age of the formulati on as a within-subje ctfixed effect .A post-hoc Tukey HSD test was used on HUMULIN® formulations to determine statistical significance between aging timepoints.

FIGs. 34A-34B illustrate the blood glucose curve for HUMULIN® including t=0.5 and t=l month time points. Fasted diabetic mal erats (n=8) received subcutaneous administrati on(1.5 U/kg) of HUMULIN®. Eight rats were randomly assigned to the HUMULIN® group. Each rat received one dose of the formulati onat each aging timepoint in a random order. Blood glucose levels were measured every 30 minutes using a handheld glucose monitor and the change in blood glucose relative to baseline glucose measurements was plotted. Statistical significance betwee nmax Aglucose was assessed using a REML repeated measures mixed model wit hrat as a random effect and the age of the formulati onas a within-subje ctfixed effect .A post-hoc Tukey HSD test was used on formulations to determine statistical significance between aging timepoints. Groups not connected by the same letter are significantly different.

FIG. 35 illustrate areas under the curve for pharmacokinetics. Area under the curve was calculated for the pharmacokinetic curves of HUMULIN®, aged HUMULIN® (t=6), HUMULIN® + M0Ni23% and aged M0Ni23% (t=6) .No difference was observed between fresh HUMULIN® or polymer stabilize dformulations Aged. HUMULIN® showed decreased area under the curve compared to HUMULIN®. All data is shown as mean ± s.e. Statistica signil ficance between AUG was assessed using a REML repeated measures mixed model wit hrat as a random effect and the age of the formulation as a within-subje ct fixed effect. A post-hoc Tukey HSD test was used on formulations to determine statistical significance betwee naging timepoints.

FIGs. 36A-36E illustrat strese sed aging in commercial packaging. FIG. 36A: HUMULIN® is often sold in standardized 10 mL glass vials and packaged in cardboard boxes. The stabilizing capacity of polyacrylamide-based copolymers in commercial packaging conditions under stresse dconditions was tested. 10 mL vial sof UI 00 HUMULIN® R were diluted to 95 U/mL wit hthe additio nof 50 pL of a M0Ni23% stock soluti on(to a final concentration of 0.01 wt.% copolymer )or water (control ).Dilution was necessary to add copolymer to the formulation. These vial swere replaced in their original boxes and taped to a shaker plat e(150 rpm) in a 37 °C (n=l per formulation) or 50 °C (n=l per formulation) incubator. Sample swere observed and imaged daily. FIGs. 36B- 36C are transmittanc assae ys for HUMULIN® or HUMULIN® comprising M0Ni23% after aging at 37 °C (FIG. 36B) and 50 °C (FIG. 36C). Single samples (n=l )were tested for each transmittanc curvee .Blood glucos curvese (FIG. 36D) and maximum change in blood glucose (A glucose) in fasted diabetic rats for samples aged at 50 °C (FIG. 36E) are shown.

Data is shown as mean ± s.e. Statistica signifl icance between max A glucos wase assessed using a REML repeated measures mixed model wit hrat as a random effect and the age of the formulation as a within-subje ctfixed effect .A post-hoc Tukey HSD test was used to determine statistical significance betwee naging timepoints and groups.

DETAILED DESCRIPTION OF EMBODIMENTS The present disclosure describe sthe synthesis and characterization of a library of copolymer scomprising different water-soluble acrylamide monomers and functional dopant monomers in a variet yof weight ratios, molecula weights,r and degree of polymerization. The copolymers of the present disclosure are effective at reducing or preventing aggregation of biologic molecules (e.g., protein and peptides) or lipid-base d vehicles in aqueous formulations at hydrophobic interfaces ,particularl moley cules that are susceptib leto aggregation in an aqueou smedium. Proteins and other biologic molecul es are often used in the treatment of a wide variet yof disease sand disorders; however, maintaining the stabilit ofy such formulations and preventing aggregation of the molecul es is a key challenge faced by the biopharmaceutical industry. Proteins, other biologic molecules, and lipid-based vehicles, including but, not limited to, liposomes, micelles , polymerosomes and, lipid nanoparticle s,can aggregate due to a variety of factors, including thermal stres s,chemical degradatio n,or exposure to surfaces and interfaces.

Non-covalent physical aggregation is mediated by forces like hydrophobic interaction, van der Waals interaction, hydrogen bonding, and electrostat forces.ic Adsorption of such molecules to a variet yof interfaces, particularl air-wy ate rinterfaces ,plays a key role in inducing aggregation. These aggregates coul dtrigge rimmunogenic responses in the body and resul int the production of anti-therapeuti antibodiesc .

The polyacrylamide-bas edcopolymers of the present disclosure have been found to reduc eor prevent aggregation of biologic molecules and lipid-based vehicles in aqueous formulations result, ing in increased stabilit y.The polyacrylamide-bas edcopolymers can be used wit hany molecule that is susceptib leto aggregation in an aqueou smedium , including but, not limited to, proteins such as antibodies and fragment sthereof, cytokines, chemokines, hormones, vaccine antigens, cancer antigens, adjuvants and, combinations thereof. In some embodiments the, protein is insulin. In some embodiments, the protein is a monoclonal antibody. Monoclonal antibodie s(mAbs) are therapeutic proteins used in the treatment of many diseases, but tend to aggregate, making their use a major challenge in formulati ondevelopment MAbs. spontaneously adsorb onto air-soluti oninterfaces and experience interfacia lstresses, which is one of the major causes of aggregation. In some embodiments, the protein is a hormone. In some embodiments, the protein is a vaccine. In some embodiments the, biologic molecule is a nuclei cacid. In some embodiments, the biologic molecule is a nuclei cacid such as mRNA, DNA, siRNA, and miRNA.

The polyacrylamide-based copolymers can also be used with lipid-based vehicle s that are susceptible to aggregation in an aqueou smedium, including but, not limited to, liposomes ,lipid nanoparticle s,polymerosomes ,and micelles to, prevent or reduce aggregation.

The polyacrylamide-based copolymers of the present disclosure prevent aggregation of proteins ,other biological molecules, and lipid-based vehicles such as liposomes li, pid nanoparticles, polymerosomes and, micelles, by providing an inert barrier at the hydrophobic interface of an aqueou sformulati onto prevent interaction between the molecules, such as protein-protein interactions. In some embodiments the, hydrophobic interface is an air-wate rinterface. In some embodiments, the hydrophobic interface is an enclosure-wat interface,er including but, not limited to, a glass-wate interface,r a rubber- water interface, a plastic-water interface, or a metal-water interface. In some embodiments , the hydrophobic interface is an oil-wat erinterface.

As described in the present disclosure copolymers, were identified that can act as stabilizing agents for formulations containing biologic molecule s.In some embodiment s, the formulati onis a protein formulatio n.The polyacrylamide-bas edcopolymers can also act as stabilizing agents for formulations containing lipid-based vehicles that are susceptib leto aggregation in an aqueous medium, including but, not limited to, liposomes , lipid nanoparticles, polymerosomes and, micelles. In some embodiments, the copolymers were identified using a high-throughpu tscreen of a large library of combinatorial acrylamide-based copolymer excipients. In some embodiments, these copolymers enhance the stabilit ofy the formulati onwithout any modifying effects on the molecule ins the formulation. For example, the copolymers can enhance protein formulati onstabilit y without any protein-modifying effects. In some embodiments, copolymers comprising a water-soluble "carrier" monomer and a functional "dopant" monomer act as stabilizing excipient sto reduce interactions of the biologic molecule wit han interface, such as the air-liquid interface. In some embodiments, the biologic molecule is a protein. In some embodiments, copolymers comprising a water-soluble "carrier" monomer and a functional "dopant" monomer act as stabilizing excipients to reduc einteractions of a lipid-base d vehicle wit han interface, such as the air-liquid interface.

Thus, provided in the present disclosure is a method for reducing aggregation of a biologic molecule or lipid-based vehicle comprising a polyacrylamide-based copolymer excipient as disclose herein.d In some embodiments, the biologic molecule is a protein. In some embodiments, the protein is selected from antibodie sand fragment sthereof, cytokines ,chemokines, hormones, vaccine antigens, cancer antigens, adjuvants and, combinations thereof. In some embodiments, the biologic molecule is a nuclei cacid. In some embodiments the, lipid-based vehicle is a liposome, micelle polymer, osome, or lipid nanoparticle. The polyacrylamide-based copolymer of the present disclosure comprises a water-soluble carrier monomer comprising an acrylamide reactive moiety and a functional dopant monomer comprising an acrylamide reactive moiety. In some embodiments, the copolymer comprises MP AM or MORPH carriers with NIP or PHE dopants.

Also provided are methods for increasing stabilit ofy a formulation containing a biologic molecule or a lipid-based vehicle .In some embodiments, methods for increasing thermal stabilit ofy a formulati oncontaining a biologic molecule or a lipid-based vehicl e are provided. In other embodiments, methods for reducing the rate of aggregation of a biologic molecule or a lipid-based vehicle in an aqueou scomposition are provided. The methods include adding the copolymer of the present disclosure to the formulation.

In some embodiments, the biologic molecule is a protein. In some embodiment s, the protein is insulin. In some embodiments, concerns of reduced insulin activity or extende dcirculation times typically associated wit hcovalent insuli nmodification (e.g., PEGylation) are reduced or eliminated.

Also provided in the present disclosure are methods of treating an elevated glucose leve lin a subject in need thereof, comprising administering to the subject a composition containing the polyacrylamide-based copolymer of the present disclosure and insulin. Also provided are methods of managing the blood glucose leve lin a subject in need thereof, comprising administering to the subject a composition containing the polyacrylamide- based copolymer of the present disclosure and insulin.

Definitions Unless otherwise defined, all technical and scientific term sused herein have the same meaning as commonl yunderstood by one of ordinary skil lin the art to which this disclosure belongs. Methods and materials are described herein for use in the present application; other, suitable methods and materials known in the art in some aspects this disclosure are also used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences , database entries, and other references mentioned herein are incorporated by reference in their entireties In. case of conflict, the present specification, including definitions, wil l control. When trade names are used herein, the trade name includes the product formulation, the generic drug, and the active pharmaceutic alingredient(s) of the trade name product, unless otherwise indicated by context.

The term "polymer" refers to a substanc eor material consisting of repeating monomer subunits.

An "acrylamide monomer," as used herein, refers to a monomer species that י* v possesses an acrylamide functional group * . The term "acrylamide monomer" includes not only monomeric acrylamide, but derivatives of monomeric acrylamide. Example sof acrylamide monomers include, but are not limited to, acrylamide (AM), N-(3-methoxypropoyl)acrylamide (MPAM), 4-acryloylmorpholine (MORPH), N,N-dimethylacrylam ide(DMA), N-hydroxy ethyl acrylamide (HEAM), N- [tris(hydroxymethyl)-methyl]acrylam (TRI)ide, 2-acrylamido-2-methylpropane sulfonic acid (AMP), (3-acrylamidopropyl)trimethylammo niumchloride (TMA), N- isopropyl acrylamide (NIP), N-tert-butyl acrylamide (TEA), and N-phenylacrylamide (PHE).

The term "polyacrylamide-based copolymer" refers to polymers that are formed from the polymerizatio nof two or more monomer species ,in which at least one of the monomer species possesses an acrylamide functional group (acrylamide monomer) and the monomers are structurally different. In some embodiments, the polyacrylamide-based copolymer is formed from the polymerization of two structurally different acrylamide monomers (two structurally different monomers that each possess an acrylamide functional group). The resulting copolymer can be an alternating copolymer wherein the monomer species are connected in an alternati ngfashion; a random copolymer, wherein the monomer species are connected to each other within a polymer chain without a defined pattern; a block copolymer, wherein polymeri cblocks of one monomer species are connected to polymeric blocks made up of another monomer species; and graft copolymer, wherein the main polymer chain consists of one monomer species ,and polymeric blocks of another monomer species are connected to the main polymer chain as side branches. In some embodiments the, polyacrylamide-bas edcopolymers of the present disclosure are formed from the polymerization of a water-soluble carrier monomer and a functional dopant monomer. In some embodiments, the polyacrylamide-bas edcopolymers of the present disclosure are random copolymers.

As defined herein, the term "water-soluble carrier monomer" refers to an acrylamide monomer species that is the water-soluble species within the polyacrylamide- based copolymer. In some embodiments, the water-soluble carrier monomer is the predominant species within the polyacrylamide-based copolymer. In some embodiment s, the water-soluble carrier monomer imparts aqueou ssolubilit toy the copolymer. In some embodiments, the water-soluble carrier monomer withi nthe polyacrylamide-based copolymer provides an inert barrier at the interface of an aqueous formulati onto prevent protein-protein interactions. In some embodiments the, interface is an air-wate rinterface.

In some embodiments, the interface is an enclosure-wate interface,r including, but not limited to, a glass-wat interface,er a rubber-wat erinterface, a plastic-water interface, or a metal-water interface. In some embodiments, the interface is an oil-wate intr erface. In some embodiments the, interface is an interface between a liquid and tubing. In some embodiments, the interface is an interface between a liqui dand a catheter .In some embodiments, the enclosure-wat interer face is in a pump system. In some embodiment s, the enclosure-wat interfaer ce is in a closed-loop system. In some embodiments the, water- solubl carrie er monomer is nonionic. Examples of water-soluble carrier monomers include, but are not limited to, acrylamide (AM), N-(3-methoxypropoyl)acrylam ide (MPAM), 4-acryloylmorpholine (MORPH), N,N-dimethylacrylam ide(DMA), and N- hydroxyethyl acrylamide (HEAM).

The term "functional dopant monomer," as used herein, refers to an acrylamide monomer species that has physicochemical properties (e.g., hydrophobicity, charge) different from those of the water-soluble carrier monomer. In some embodiments the, functional dopant monomer within the polyacrylamide-based copolymer promotes association of the polymers to an interface; such interfaces can include, but are not limited to, polymer-air-wat erinterface interactions, polymer-protein interactions, polymer- peptide interactions, polymer-micelle interactions, polymer-liposom interae ctions and, polymer-lipid nanoparticle interactions. The functional dopant monomer can act as a stabilizing moiety to facilitate interactions with biomolecules, for example, proteins, peptides, antibodies, antibody-drug conjugates, nuclei c acids, lipid particles ,and combinations thereof (e.g., to prevent aggregation of the biomolecules The). functional dopant monomers can be further classified into hydrogen-bonding, ionic, hydrophobic, and aromati cmonomers based on their chemical composition. Typically, the functional dopant monomers are copolymerized at a lower weight percentages as compared to the water-soluble carrier monomers.

The term "polymerizatio"n refers to the process in which monomer molecul es undergo a chemical reaction to form polymeric chains or three-dimensional networks .

Different types of polymerizatio nreactions are known in the art, for example, addition (chain-reaction) polymerization, condensation polymerization, ring-opening polymerization, free radical polymerization, controll edradical polymerization, atom transfer radical polymerization (ATRP), single-electron transfer living radical polymerization (SET-LRP), reversibl eaddition-fragmentation chain transfer (RAFT) polymerization, nitroxide-mediate polymd erizatio (NMP),n and emulsion polymerization.

In some embodiments, the copolymers of the present disclosure are prepared using RAFT polymerization.

The term "degree of polymerization" (DP) refers to the number of monomer unit s in a polymer. It is calculated by dividing the average molecula weir ght of a polymer sampl e by the molecula weir ght of the monomers. As defined herein, the average molecula r weight of a polymer can be represented by the number-averaged molecula weir ght (Mn), the weight-average molecula weighr t (Mw), the Z-average molecula weir ght (Mz) or the molecula weir ght at the peak maxima of the molecula weir ght distribut ioncurve (Mp). The average molecul arweight of a polymer can be determined by a variety of analytica l characterization techniques known to those skilled in the art, for example, gel permeation chromatography (GPC), static light scattering (SLS) analysis ,multi-angl lase er light scattering (MALLS) analysis nucle, ar magnetic resonance spectroscopy (NMR), intrinsi c viscomet ry(IV), melt flow index (MFI), and matrix-assist edlaser desorption/ionization mass spectrometry (MALDI-MS), and combinations thereof. Degree of polymerization can also be determined experimentall usingy suitable analytical methods known in the art, such as nuclea rmagnetic spectroscopy (NMR), Fourier Transform infrared spectroscopy (FT-IR) and Raman spectroscopy.

The term "amphiphilic" refers to chemical substance thats posses sboth hydrophilic (water-loving, polar) and lipophilic (fat-loving, nonpolar) properties .Example sof common amphiphilic compounds include detergents, soaps, surfactants, lipoproteins, and phospholipids. In some embodiments, the amphiphilic substance is a charged species .In some embodiments the, amphiphilic substance is a neutra lspecies.

A "lipid-based vehicle," as used herein, refers to structures having a protective outer layer of lipids that can be used as drug delivery vehicles. For example, a lipid-base d vehicle can be used to encapsula teand transport cargo (e.g., a therapeutic agent) to a biologic target Example. sof lipid-based vehicle sinclude, but are not limited to, liposomes , micelles polymer, osomes and, lipid nanoparticles.

"Biologic molecul"e, as used herein, refers to molecules such as proteins ,nucleic acids, polysaccharides and, lipids.

The term "protein" is defined as a clas sof large molecules comprising one or more long chains of amino acids .A wide variety of proteins may be considered as belonging to a family of proteins based on having similar structural features, having particular biological functions, and/or being related to specific microorganisms, particularl disey ase causing microorganisms. Such proteins include, for example, antibodies (immunoglobulins), cytokines ,chemokines, enzymes, hormones, vaccine antigens, cancer antigens, adjuvants, nutritional markers, and tissue specific antigens.

The term "nuclei cacid," as used herein, includes deoxyribonuclei cacid (DNA), ribonucleic acid (RNA), messenger RNA (mRNA), small-interfering RNA (siRNA), short hairpin RNA (shRNA), and microRNA (miRNA).

The term "antibody" refers to large, Y-shaped proteins produced by the immune system to identify and neutrali zeforeign objects such as pathogenic bacteria and viruses.

The term "antibody" includes monoclonal antibodies (for example, full length or intact monoclonal antibodies) ,polyclonal antibodies, multivalent antibodies, multispecif ic antibodies (e.g., bispecific or trispecific antibodies, so long as they exhibit the desired biological activity) and can also include certain antibody fragments .An antibody can be human, humanized and/or affinity matured. "Antibody fragments" comprise only a portion of an intact antibody, where in certain embodiments, the portion retains at least one, and typically most or all of, the functions normally associated wit hthat portion when present in an intact antibody. In one embodiment an, antibody fragment comprises an antigen binding sit eof the intact antibody and thus retains the ability to bind antigen. In another embodiment, an antibody fragment, for example one that comprises the Fc region, retains at least one of the biological functions normally associate dwit hthe Fc region when present in an intact antibody, such as FcRn binding, antibody half-life modulation, ADCC function and complement binding. In one embodiment, an antibody fragment is a monovalent antibody that has an in vivo half-life substantiall simy ilar to an intact antibody. For example, such an antibody fragment may comprise an antigen binding arm linked to an Fc sequence capable of conferring in vivo stabilit toy the fragment.

The term "monoclona lantibody" as used herein refers to an antibody obtained from a populatio nof substantia llyhomogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possibl enatural lyoccurring mutatio ns that may be present in minor amounts. Monoclonal antibodie sare highly specific, being directed against a singl e antigen. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes ),each monoclonal antibody is directed against a singl edeterminant on the antigen.

The monoclonal antibodies herein include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical wit hor homologous to corresponding sequences in antibodie sderived from a particular species or belonging to a particular antibody clas s or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody clas sor subclass, as wel las fragment sof such antibodies, so long as they exhibit the desired biological activity.

The term "insuli"n refers to a hormone produced by the beta cell ins the pancreatic islets that regulat esthe amount of glucos ein the blood. Many eukaryotes ,including humans ,primates, pigs, cows, cats, dogs, and rodents, produce insulin. Thus, "insulin," as used herein, includes insulin produced by humans ,and analogs thereof, as wel las insulin, and analogs thereof, produced by other eukaryotes including,, but not limited to, primates , pigs, cows ,cats, dogs, and rodents, and also includes recombinant, purified or synthet ic insulin or insulin analogs having simila functr ion and structure, unless otherwise specified .

The human insuli nprotein consists of 51 amino acids ,and has a molecul arweight of approximatel 5.8y kilodalton (kDa). Human insulin is a heterodime ofr an A-chain and a B-chain that are connected by disulfide bonds.

Insuli nalso includes monomeric and oligomeri cforms, such as dimeric and hexameric forms .Insuli ncan exist as a monomer as it circulates in the plasma, and it also binds to its receptor while in a monomeric form. Insuli nformulations (or insulin analog formulations) containing a predominance of protein molecules in the form of monomers and dimers ordinarily have a stron gtendenc yto aggregate and form inactive fibrils. Insuli n hexamers are too large to be absorbed, and so hexameric insulin formulations mus t disassembl inte o dimers or monomers before the insuli ncan be absorbed and function in the body. The active form of insulin in the blood stream is the monomeric form.

Insuli ncan be isolated from the pancreatic islets extracts of an animal that produces insulin, or expressed recombinantl yin a suitabl expresse ion system such as E. coli, yeast , insect cell s,and mammalian cell s(e.g. Chinese hamster ovary (CHO) cells ).Depending upon their specific pharmacokinetics and pharmacodynamics (PK/PD) properties (e.g. duration of action, maximum concentration observed (Cmax), time-to-onset, area under the curve (AUC)), insulin can be further characterized as a rapid-acting insulin, a short-acting insulin, an intermediate-act inginsulin, a long-acting insulin, and a pre-mixed insulin.

The term "aggregation" refers to the formation of higher molecula weir ght, amorphous species due to non-covalent adherence ("clumpin"g) of smalle specir es. The aggregation process can be irreversible or reversibl e.Many biological and synthetic molecules can undergo aggregation, including proteins ,peptides, lipid particles nucleic, acids, inorganic nanoparticles and organic nanoparticle s(e.g., micelles ,lipid nanoparticles, liposomes polymeroso, mes) that may furthe rcomprise an encapsulat ed species.

In the case of protein aggregation, formation of protein aggregates can be due to the protein’s intrinsic disordere dnature, or misfolding of protein molecules, which result s in the exposure of hydrophobic residues and surfaces that are normally buried within the interior of the protein three-dimensional structure. Due to the hydrophobic effect , the exposed hydrophobic portions of a misfolded protein have the tendenc yto interact wit h other misfolded protein molecules to shield the exposed hydrophobic surfaces, which can lead to protein aggregation.

Some biologic molecul esare more "susceptible to aggregation" than others .For example, the amino-acid sequence and overall three-dimensional structure of a protein is relevant to its susceptibil toity aggregation. For example, transmembrane proteins are more prone (or susceptible) to aggregation than non-membrane proteins ,particularly when expressed recombinantl wity hout the use of a stabilizing agent. Proteins that are subject to conditions beyond the physiological conditions (37 °C, ~ neutral pH, isotonic )may also be more susceptible to aggregation than when in their native environment .Stres s conditions such as temperatur flucte uations ligh, t, mechanical perturbation (e.g., shaking), surfaces, ultrasonic vibration, pH changes, and changes in ionic strength can affect protein stabilit andy induce aggregation. Protein aggregation can lead to the formation of sub- visible or visible particles (i.e., precipitation). The extent of sub-visible protein aggregation can be measured by a variet yof analytical methods known in the art, for example, size-exclusion chromatography (SEC), gel electrophoresis, asymmetric field- flow fractionation (AF4), analytica ultral centrifugati (AUCon ), mass spectrometry (MS), optical microscopy, fluorescence microscopy, dynamic light scattering (DLS), multi-angle laser light scattering (MALLS), flow imaging, turbidity/nephelomet andry, transmittance measurement.

As used herein, the term "reduced aggregation" of a biologic molecule or lipid - based vehicl eincludes all forms of reducing aggregation. The degree or amount of aggregation observed (e.g., in the composition) can be reduced as compared to a composition of the same biologic molecule or lipid-based vehicle in the absence of the polyacrylamide-bas edcopolymer of the present disclosure. Thus, "reduced aggregation" includes no observabl eaggregation or reduced amounts of aggregation (e.g., reduced levels of aggregated protein). Thus, the amount of aggregates present in the composition can be reduced by at least about 10 mol%, about 20 mol% ,about 30 mol% ,about 40 mol%, about 50 mol%, about 60 mol% ,about 70 mol%, about 80 mol%, about 90 mol%, or about 100 mol % as compared to the amount of aggregates of the same biologic molecule or lipid - based vehicle in the absence of the polyacrylamide-based copolymer. Aggregation can be measured by any method known in the art, including but, not limited to, size-exclusion chromatography (SEC), gel electrophoresi s,asymmetric field-flow fractionation (AF4), analytical ultracentrifugat ion(AUC), mass spectromet ry(MS), optical microscopy, fluorescence microscopy, dynamic light scattering (DLS), multi-angl lase er light scattering (MALLS), flow imaging, turbidity/nephelomet ry,and transmittance measurement.

As used herein, the term "increased stability" when, referring to a formulati on containing a biologic molecule or lipid-based vehicle ,refers to a measurabl decree ase in the amount of aggregation over a fixed period of time under testing or fixed storage conditions as compared to the amount of aggregates of the same biologic molecule or lipid - based vehicle in the absence of the polyacrylamide-based copolymer.

The term s"aggregated protein" or "protein aggregates" as used herein refer to a collection of proteins that are disordere dor misfolded and grouped together. The aggregates can be solubl ore insolubl Proteie. n aggregates include, but are not limited to, inclusion bodies, soluble and insoluble precipitate s,soluble non-native oligomers ,gels, fibrils, films, filaments, protofibrils, amyloid deposits amyl, oid fibrils, plaques, and disperse dnon-native intracellul oligomersar .In some embodiments, the proteins in a protein aggregate are, prior to their aggregation, solubl precursore s. Protein aggregation can be prevented in compositions containing the polyacrylamide-bas edcopolymer of the present disclosure. Protein aggregation can also be reduced in a composition containing the polyacrylamide-based copolymer of the present disclosure as compared to a composition containing the same protein that does not contain the polyacrylamide-based copolymer of the present disclosure. Thus, the polyacrylamide-based copolymer can reduce or prevent the aggregation of a protein.

In some embodiments the, protein is insulin, or an analog thereof. The term s "aggregated insulin" or "insuli naggregates" as used herein, refer to insuli nthat has aggregated to form a high molecul arweight polymer or aggregate particles or amyloid fibrils. Formation of insuli naggregates can be due to, for example, heat or shaking and partial unfolding of the insulin. Insuli naggregation can be prevented, i.e., insuli n molecules are prevented from aggregating and forming high molecula weir ght polymers or amyloid fibrils, in compositions containing the polyacrylamide-bas edcopolymer of the present disclosure. Aggregation can also be reduced in a composition containing insulin, or an analog thereof, and the polyacrylamide-based copolymer of the present disclosure as compared to a composition containing insulin, or an analog thereof, that does not contain the polyacrylamide-bas edcopolymer of the present disclosure. Thus, the polyacrylamide- based copolymer can reduce or prevent the aggregation of insulin, or analogs thereof.

As defined herein, the term "duration of action" is defined as the lengt hof tim e that injected insulin is acting to lower blood glucose level s,i.e., the timeframe over which the injected insulin continues to be active. The term "maximum concentration observed" (Cmax) refers to the peak serum concentration that an active agent achieves in a specified compartmen tor test area of the body after administration of the first dose of the active agent and before the administration of the second dose. The term "time-to-onset" refers to the amount of time it takes an active agent to reach the minimum effective concentration after administration. The term "area under the curve" (AUC) refers to the definite integral of a curve that describes the variation of the concentration of the active agent in blood plasm aas a function of time, which can be measured by analytical methods such as liquid- chromatography-mass spectromet ry(LC-MS). The AUC reflects the exposure of the active agent upon administration, and is expressed in mg*h/L. The AUC of an active agent is dependent on the rate of elimination of the active agent from the body and the dose administered.

Polyacrylamide-based copolymers Provided in the present disclosure are polyacrylamide-based copolymers In. some embodiments, the polyacrylamide-based copolymers contain a water-soluble carrier monomer and a functional dopant monomer. In some embodiments, the polyacrylamide- based copolymer is amphiphilic.

In some embodiments the, polyacrylamide-bas copolymered comprises a non-ionic water-soluble acrylamide monomer and a functional acrylamide dopant monomer selected from the group consisting of a hydrophobic functional acrylamide dopant monomer, an aromati c functional acrylamide dopant monomer, a hydrogen-bonding functional acrylamide dopant monomer, and an ionic functional acrylamide dopant monomer. In some embodiments, the polyacrylamide-bas edcopolymer comprises a non-ionic water- solubl eacrylamide monomer and the functional acrylamide dopant monomer is a hydrophobic functional acrylamide dopant monomer. In some embodiments, the copolymer comprises a non-ionic water-soluble acrylamide monomer and the functional acrylamide dopant monomer is an aromati cfunctional acrylamide dopant monomer. In some embodiments the, copolymer comprises a non-ionic water-soluble acrylamide monomer and the functional acrylamide dopant monomer is a hydrogen-bonding functional acrylamide dopant monomer. In some embodiments, the copolymer comprises a non-ionic water-soluble acrylamide monomer and the functional acrylamide dopant monomer is an ionic functional acrylamide dopant monomer.

The polyacrylamide-bas edcopolymers of the present disclosure contain a water- solubl carrie er monomer. In some embodiments the, water-soluble carrier monomer is non-ionic. In some embodiments the, water-soluble carrier monomer is selected from the group consisting of 7V-(3-methoxypropoyl)acrylamide (MPAM), 4-acryloylmorpholine (MORPH), 7V,7V-dimethylacrylami (DMAde ), 7V-hydroxyethyl acrylamide (HEAM), and acrylamide (AM), or combinations thereof. In some embodiments the, water-solubl e carrier monomer is selected from the group consisting of MP AM and MORPH. In some embodiments, the water-soluble carrier monomer is 7V-(3-methoxypropoyl)acrylami de (MPAM). In some embodiments, the water-soluble carrier monomer is 4- acryloylmorpholine (MORPH). In some embodiments, the water-soluble carrier monomer is 7V,7V-dimethylacrylami de(DMA). In some embodiments the, water-soluble carrier monomer is 7V-hydroxyethyl acrylamide (HEAM). In some embodiments the, water- solubl carrie er monomer is acrylamide (AM). In some embodiments the, copolymer comprises a water-soluble carrier monomer selected from the group consisting of A-(3- methoxypropoyl)acrylam ide(MPAM) and 4-acryloylmorpholine (MORPH).

The polyacrylamide-based copolymers of the present disclosure also contain a functional dopant monomer. In some embodiments the, functional dopant monomer is selected from the group consisting of a hydrophobic functional acrylamide dopant monomer, an aromati cfunctional acrylamide dopant monomer, a hydrogen-bonding functional acrylamide dopant monomer, and an ionic functional acrylamide dopant monomer, or mixture thers eof.

In some embodiments, the functional acrylamide dopant monomer is a hydrophobic functional acrylamide dopant monomer. In some embodiments, the hydrophobic functional acrylamide dopant monomer is 7V-isopropylacrylamide (NIP) or 7V-tert-butyl acrylamide (TEA). In some embodiments, the hydrophobic functional acrylamide dopant monomer is N-isopropyl acrylamide (NIP). In some embodiments, the hydrophobic functional acrylamide dopant monomer is 7V-/ert-butylacrylam (TEAide ). In some embodiments, the functional acrylamide dopant monomer is an aromati cfunctional acrylamide dopant monomer. In some embodiments, the aromati cfunctional acrylamide dopant monomer is 7V-phenyl acrylamide (PHE).

In some embodiments, the functional acrylamide dopant monomer is a hydrogen- bonding functional acrylamide dopant monomer. In some embodiments, the hydrogen- bonding functional acrylamide dopant monomer is 7V-[tris(hydroxymethyl)- methyl]acrylamide (TRI). In some embodiments, the functional acrylamide dopant monomer is an ionic functional acrylamide dopant monomer. In some embodiments, the ionic functional acrylamide dopant monomer is 2-acrylamido-2-methylpropane sulfonic acid (AMP) or (3-acrylamidopropyl)trimethylammonium chloride (TMA). In some embodiments, the ionic functional acrylamide dopant monomer is 2-acrylamido-2- methylpropane sulfonic acid (AMP). In some embodiments, the ionic functional acrylamide dopant monomer is (3-acrylamidopropyl)trimethylammo niumchloride (TMA).

In some embodiments, the functional dopant monomer is selected from the group consisting of N-[tris(hydroxymethyl)-methyl]acrylam (TRI)ide , 2-acrylamido-2- methylpropane sulfonic acid (AMP), (3-acrylamidopropyl)trimethylammo chloridenium (TMA), N-isopropylacrylamide (NIP), N-tert-butyl acrylamide (TBA), and N- phenyl acrylamide (PHE), or combinations thereof. In some embodiments, the functional dopant monomer is N-[tris(hydroxymethyl)-methyl]acrylam (TRI)ide . In some embodiments, the functional dopant monomer is 2-acrylamido-2-methylpropane sulfonic acid (AMP). In some embodiments ,the functional dopant monomer is (3- acrylamidopropyl)trimethylammonium chloride (TMA). In some embodiments, the functional dopant monomer is N-isopropylacrylamide (NIP). In some embodiments, the functional dopant monomer is N-tert-butylacrylamide (TBA). In some embodiments the, functional dopant monomer is and N-phenylacrylamide (PHE).

In some embodiments the, polyacrylamide-base copolymerd comprises a water- soluble carrier monomer selected from the group consisting of N-(3- methoxypropoyl)acrylam ide(MPAM), 4-acryloylmorpholine (MORPH), N,N- dimethylacrylamide (DMA), N-hydroxy ethyl acrylamide (HEAM), and acrylamide (AM); and a functional dopant monomer selected from the group consisting of N- [tris(hydroxymethyl)-methyl]acrylam (TRI)ide, 2-acrylamido-2-methylpropane sulfonic acid (AMP), (3-acrylamidopropyl)trimethylammo niumchloride (TMA), N- isopropyl acrylamide (NIP), N-tert-butylacrylami (TBA),de and N-phenylacrylamide (PHE).

In some embodiments, the water-soluble carrier monomer is N-(3- methoxypropoyl)acrylam ide(MPAM). In some embodiments, the water-soluble carrier monomer is N-(3-methoxypropoyl)acrylamide (MPAM) and the functional dopant monomer selected from the group consisting of N-[tris(hydroxymethyl)- methyl]acrylamide (TRI), 2-acrylamido-2-methylpropane sulfoni cacid (AMP), (3- acrylamidopropyl)trimethylammoniu chloridem (TMA), N-isopropylacrylamide (NIP), N-tert-butylacrylam (TBA),ide and N-phenylacrylamide (PHE). In some embodiments , the water-soluble carrier monomer is MPAM and the functional dopant monomer is TRI.

In some embodiments the, water-soluble carrier monomer is MPAM and the functional dopant monomer is AMP. In some embodiments the, water-soluble carrier monomer is MPAM and the functional dopant monomer is TMA. In some embodiments, the water- solubl carrie er monomer is MPAM and the functional dopant monomer is NIP. In some embodiments, the water-soluble carrier monomer is MPAM and the functional dopant monomer is TBA. In some embodiments the, water-soluble carrier monomer is MPAM and the functional dopant monomer is PHE.

In some embodiments, the water-soluble carrier monomer is 4-acryloylmorpholine (MORPH). In some embodiments ,the water-soluble carrier monomer is 4- acryloylmorpholine (MORPH) and the functional dopant monomer selected from the group consisting of N-[tris(hydroxymethyl)-methyl]acryl amide(TRI), 2-acrylamido-2- methylpropane sulfonic acid (AMP), (3-acrylamidopropyl)trimethylammo chloridenium (TMA), N-isopropylacrylami de(NIP), N-tert-buty acrlylamide (TEA), and N- phenyl acrylamide (PHE). In some embodiments, the water-soluble carrier monomer is MORPH and the functional dopant monomer is TRI. In some embodiments, the water- solubl carrie er monomer is MORPH and the functional dopant monomer is AMP. In some embodiments, the water-soluble carrier monomer is MORPH and the functional dopant monomer is TMA. In some embodiments, the water-soluble carrier monomer is MORPH and the functional dopant monomer is NIP. In some embodiments, the water-solubl e carrier monomer is MORPH and the functional dopant monomer is TEA. In some embodiments, the water-soluble carrier monomer is MORPH and the functional dopant monomer is PHE.

In some embodiments , the water-soluble carrier monomer is N,N- dimethylacrylam ide(DMA). In some embodiments, the water-soluble carrier monomer is N,N-dimethylacrylam ide(DMA) and the functional dopant monomer selected from the group consisting of N-[tris(hydroxymethyl)-methyl]acryl amide(TRI), 2-acrylamido-2- methylpropane sulfonic acid (AMP), (3-acrylamidopropyl)trimethylammo chloridenium (TMA), N-isopropylacrylamide (NIP), N-tert-butyl acrylamide (TEA), and N- phenyl acrylamide (PHE). In some embodiments, the water-soluble carrier monomer is DMA and the functional dopant monomer is TRI. In some embodiments, the water-solubl e carrier monomer is DMA and the functional dopant monomer is AMP. In some embodiments, the water-soluble carrier monomer is DMA and the functional dopant monomer is TMA. In some embodiments, the water-soluble carrier monomer is DMA and the functional dopant monomer is NIP. In some embodiments the, water-soluble carrier monomer is DMA and the functional dopant monomer is TEA. In some embodiments the, water-soluble carrier monomer is DMA and the functional dopant monomer is PHE.

In some embodiments, the water-soluble carrier monomer is N-hydroxyethyl acrylamide (HEAM). In some embodiments, the water-solubl carrie er monomer is N- hydroxyethyl acrylamide (HEAM) and the functional dopant monomer selected from the group consisting of N-[tris(hydroxymethyl)-methyl]acryl amide(TRI), 2-acrylamido-2- methylpropane sulfonic acid (AMP), (3-acrylamidopropyl)trimethylammo chloridenium (TMA), N-isopropylacrylami de(NIP), N-tert-butyl acrylamide (TBA), and N- phenyl acrylamide (PHE). In some embodiments, the water-soluble carrier monomer is HEAM and the functional dopant monomer is TRI. In some embodiments, the water- solubl carrie er monomer is HEAM and the functional dopant monomer is AMP. In some embodiments, the water-soluble carrier monomer is HEAM and the functional dopant monomer is TMA. In some embodiments, the water-soluble carrier monomer is HEAM and the functional dopant monomer is NIP. In some embodiments, the water-solubl e carrier monomer is HEAM and the functional dopant monomer is TBA. In some embodiments, the water-soluble carrier monomer is HEAM and the functional dopant monomer is PHE.

In some embodiments, the water-soluble carrier monomer is acrylamide (AM). In some embodiments, the water-soluble carrier monomer is acrylamide (AM) and the functional dopant monomer selected from the group consisting of N-[tris(hydroxymethyl)- methyl]acrylamide (TRI), 2-acrylamido-2-methylpropane sulfoni cacid (AMP), (3- acrylamidopropyl)trimethylammoniu chloridem (TMA), N-isopropylacrylamide (NIP), N-tert-butylacrylam (TBA),ide and N-phenylacrylamide (PHE). In some embodiments , the water-soluble carrier monomer is AM and the functional dopant monomer is TRI. In some embodiments, the water-soluble carrier monomer is AM and the functional dopant monomer is AMP. In some embodiments the, water-soluble carrier monomer is AM and the functional dopant monomer is TMA. In some embodiments, the water-solubl carre ier monomer is AM and the functional dopant monomer is NIP. In some embodiments, the water-soluble carrier monomer is AM and the functional dopant monomer is TBA. In some embodiments, the water-soluble carrier monomer is AM and the functional dopant monomer is PHE.

In some embodiments the, functional dopant monomer is N-[tris(hydroxymethyl)- methyl]acrylamide (TRI). In some embodiments, the functional dopant monomer is N- [tris(hydroxymethyl)-methyl]acryl amide(TRI) and the water-soluble carrier monomer selected from the group consisting of N-(3-methoxypropoyl)acrylamide (MPAM), 4- acryloylmorpholine (MORPH), N,N-dimethylacrylam ide(DMA), N-hydroxyethyl acrylamide (HEAM), and acrylamide (AM). In some embodiments the, functional dopant monomer is TRI and the water-soluble carrier monomer is MP AM. In some embodiments , the functional dopant monomer is TRI and the water-soluble carrier monomer is MORPH.

In some embodiments, the functional dopant monomer is TRI and the water-soluble carrier monomer is DMA. In some embodiments, the functional dopant monomer is TRI and the water-soluble carrier monomer is HEAM. In some embodiments the, functional dopant monomer is TRI and the water-soluble carrier monomer is AM.

In some embodiments ,the functional dopant monomer is 2-acrylamido-2- methylpropane sulfonic acid (AMP). In some embodiments, the functional dopant monomer is 2-acrylamido-2-methylpropane sulfoni cacid (AMP) and the water-solubl e carrier monomer selected from the group consisting of N-(3-methoxypropoyl)acrylam ide (MPAM), 4-acryloylmorpholine (MORPH), N,N-dimethylacrylam ide(DMA), N- hydroxyethyl acrylamide (HEAM), and acrylamide (AM). In some embodiments, the functional dopant monomer is AMP and the water-soluble carrier monomer is MPAM. In some embodiments the, functional dopant monomer is AMP and the water-soluble carrier monomer is MORPH. In some embodiments, the functional dopant monomer is AMP and the water-soluble carrier monomer is DMA. In some embodiments, the functional dopant monomer is AMP and the water-solubl carrie er monomer is HEAM. In some embodiments, the functional dopant monomer is AMP and the water-soluble carrier monomer is AM.

In some embodiments, the functional dopant monomer is (3- acrylamidopropyl)trimethylammonium chloride (TMA). In some embodiments, the functional dopant monomer is (3-acrylamidopropyl)trimethylammonium chloride (TMA) and the water-soluble carrier monomer selected from the group consisting of N-(3- methoxypropoyl)acrylam ide(MPAM), 4-acryloylmorpholine (MORPH), N,N- dimethylacrylamide (DMA), N-hydroxyethyl acrylamide (HEAM), and acrylamide (AM).

In some embodiments, the functional dopant monomer is TMA and the water-solubl e carrier monomer is MPAM. In some embodiments the, functional dopant monomer is TMA and the water-solubl carrie er monomer is MORPH. In some embodiments the, functional dopant monomer is TMA and the water-soluble carrier monomer is DMA. In some embodiments the, functional dopant monomer is TMA and the water-soluble carrier monomer is HEAM. In some embodiments, the functional dopant monomer is TMA and the water-soluble carrier monomer is AM.

In some embodiments the, functional dopant monomer is N-isopropylacrylamide (NIP). In some embodiments, the functional dopant monomer is N-isopropylacrylami de (NIP) and the water-soluble carrier monomer selected from the group consisting of N-(3- methoxypropoyl)acrylam ide(MPAM), 4-acryloylmorpholine (MORPH), N,N- dimethylacrylamide (DMA), N-hydroxy ethyl acrylamide (HEAM), and acrylamide (AM).

In some embodiments, the functional dopant monomer is NIP and the water-soluble carrier monomer is MPAM. In some embodiments the, functional dopant monomer is NIP and the water-soluble carrier monomer is MORPH. In some embodiments the, functional dopant monomer is NIP and the water-soluble carrier monomer is DMA. In some embodiments, the functional dopant monomer is NIP and the water-soluble carrier monomer is HEAM. In some embodiments, the functional dopant monomer is NIP and the water-soluble carrier monomer is AM.

In some embodiments, the functional dopant monomer is N-tert-butylacrylamide (TEA). In some embodiments, the functional dopant monomer is N-tert-butylacrylam ide (TEA) and the water-soluble carrier monomer selected from the group consisting of N-(3- methoxypropoyl)acrylam ide(MPAM), 4-acryloylmorpholine (MORPH), N,N- dimethylacrylamide (DMA), N-hydroxyethyl acrylamide (HEAM), and acrylamide (AM).

In some embodiments, the functional dopant monomer is TEA and the water-solubl e carrier monomer is MPAM. In some embodiments the, functional dopant monomer is TEA and the water-soluble carrier monomer is MORPH. In some embodiments, the functional dopant monomer is TEA and the water-soluble carrier monomer is DMA. In some embodiments, the functional dopant monomer is TEA and the water-soluble carrier monomer is HEAM. In some embodiments the, functional dopant monomer is TEA and the water-soluble carrier monomer is AM.

In some embodiments, the functional dopant monomer is N-phenylacrylamide (PHE). In some embodiments, the functional dopant monomer is N-phenylacrylamide (PHE) and the water-soluble carrier monomer selected from the group consisting of N-(3- methoxypropoyl)acrylam ide(MPAM), 4-acryloylmorpholine (MORPH), N,N- dimethylacrylamide (DMA), N-hydroxyethyl acrylamide (HEAM), and acrylamide (AM).

In some embodiments, the functional dopant monomer is PHE and the water-solubl e carrier monomer is MPAM. In some embodiments, the functional dopant monomer is PHE and the water-soluble carrier monomer is MORPH. In some embodiments, the functional dopant monomer is PHE and the water-soluble carrier monomer is DMA. In some embodiments, the functional dopant monomer is PHE and the water-soluble carrier monomer is HEAM. In some embodiments, the functional dopant monomer is PHE and the water-soluble carrier monomer is AM.

In some embodiments, the polyacrylamide-bas edcopolymer comprises A-(3- methoxypropoyl)acrylam ide(MPAM) or 4-acryloylmorpholine (MORPH) as the water- solubl carrie er monomer, andN-isopropylacrylami (NIP)de or 7V-phenyl acrylamide (PHE) as the functional dopant monomer. In some embodiments, the polyacrylamide-based copolymer comprises 7V,7V-dimethylacrylami de(DMA), 7V-hydroxyethyl acrylamide (HEAM), or acrylamide (AM) as the water-soluble carrier monomer, and N- isopropyl acrylamide (NIP) or 7V-phenyl acrylamide (PHE) as the functional dopant monomer.

In some embodiments, the polyacrylamide-bas edcopolymer comprises A-(3- methoxypropoyl)acrylam ide(MPAM) or 4-acryloylmorpholine (MORPH) as the water- solubl ecarrier monomer, and 7V-[tris(hydroxymethyl)-methyl]acryla mide(TRI), 2- acrylamido-2-m ethylpropane sulfoni c acid (AMP), (3- acrylamidopropyl)trimethylammonium chloride (TMA), or /V-Zc/7-butylacrylam ide (TEA) as the functional dopant monomer.

In some embodiments, the polyacrylamide-bas edcopolymer comprises N,N- dimethylacrylamide (DMA), N-hydroxyethyl acrylamide (HEAM), or acrylamide (AM) as the water-soluble carrier monomer, and the functional dopant monomer is (3- acrylamidopropyl)trimethylammoniu chlomride (TMA). In some embodiments, the polyacrylamide-bas edcopolymer comprises 7V,7V-dimethylacrylami de(DMA), N- hydroxyethyl acrylamide (HEAM), or acrylamide (AM) as the water-soluble carrier monomer, and the functional dopant monomer is 7V-/er/-butylacrylamide (TEA). In some embodiments, the polyacrylamide-based copolymer comprises 7V,7V-dimethylacrylami de (DMA), 7V-hydroxyethyl acrylamide (HEAM), or acrylamide (AM) as the water-solubl e carrier monomer, and the functional dopant monomer is 7V-[tris(hydroxymethyl )- methyl]acrylamide (TRI). In some embodiments the, polyacrylamide-based copolymer comprises 7V,7V-dimethylacrylami de(DMA), 7V-hydroxyethyl acrylamide (HEAM), or acrylamide (AM) as the water-soluble carrier monomer, and the functional dopant monomer is 2-acrylamido-2-methylpropane sulfonic acid (AMP).

In some embodiments, the amount of functional dopant monomer used in the copolymerization reaction is designed to maximize dopant loading whil eyielding functional copolymers wit hlower critica lsoluti ontemperature (LCST) values above 37°C.

In some embodiments, this results in copolymers that remain soluble at all relevant temperatures. In some embodiments the, polyacrylamide-based copolymer comprises about 2% to about 30% by weight of a functional dopant monomer, for example, about 5% to about 30%, about 10% to about 30%, about 15% to about 30%, about 20% to about %, about 25% to about 30%, about 2% to about 25%, about 5% to about 25%, about % to about 25%, about 15% to about 25%, about 20% to about 25%, about 2% to about 20%, about 5% to about 20%, about 10% to about 20%, about 15% to about 20%, about 2% to about 15%, about 5% to about 15%, about 10% to about 15%, about 2% to about %, about 5% to about 10%, or about 2% to about 5%, by weight of a functional dopant monomer. In some embodiments, the polyacrylamide-bas edcopolymer comprises about 2%, about 5%, about 8%, about 10%, about 12%, about 15%, about 18%, about 20%, about 22%, about 25%, about 28%, or about 30% by weight of a functional dopant monomer.

In some embodiments, the polyacrylamide-bas copolymed er comprises about 70% to about 98% by weight of a water-soluble carrier monomer, for example, about 75% to about 98%, about 80% to about 98%, about 85% to about 98%, about 90% to about 98%, about 95% to about 98%, about 70% to about 95%, about 75% to about 95%, about 80% to about 95%, about 85% to about 95%, about 90% to about 95%, about 70% to about 90%, about 75% to about 90%, about 80% to about 90%, about 85% to about 90%, about 70% to about 85%, about 75% to about 85%, about 80% to about 85%, about 70% to about 80%, about 75% to about 80%, or about 70% to about 75%, by weight of a water-solubl e carrier monomer. In some embodiments the, polyacrylamide-based copolymer comprises about 70%, about 72%, about 75%, about 78%, about 80%, about 82%, about 85%, about 88%, about 90%, about 92%, about 95%, or about 98% by weight of a water-soluble carrier monomer.

In some embodiments, the polyacrylamide-bas copolymed er comprises about 70% to about 98% by weight of a water-soluble carrier monomer and about 2% to about 30% by weight of a functional dopant monomer. For example, the polyacrylamide-based copolymer can contain about 70% to about 98%, about 70% to about 95%, about 70% to about 80%, about 80% to about 90%, about 90% to about 98%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 98% by weight of a water-solubl e carrier monomer and about 2% to about 30%, about 5% to about 25%, about 5% to about %, about 2% to about 5%, about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 25%, or about 25% to about 30%, about 2%, about 5%, about 8%, about 10%, about 12%, about 15%, about 18%, about 20%, about 22%, about %, about 28%, or about 30% by weight of a functional dopant monomer.

In some embodiments the, polyacrylamides-based copolymer comprises about 2% to about 30% by weight of the functional dopant monomer NIP. In some embodiment s, the polyacrylamides-based copolymer comprises about 5% to about 30% by weight of the functional dopant monomer NIP. In some embodiments the, polyacrylamides-based copolymer comprises about 10% to about 28% by weight of the functional dopant monomer NIP. In some embodiments, the polyacrylamides-based copolymer comprises about 5% to about 26% by weight of the functional dopant monomer NIP. In some embodiments, the polyacrylamides-based copolymer comprises about 5% to about 10% by weight of the functional dopant monomer NIP. In some embodiments, the polyacrylamides-based copolymer comprises about 10% to about 15% by weight of the functional dopant monomer NIP. In some embodiments the, polyacrylamides-based copolymer comprises about 15% to about 20% by weight of the functional dopant monomer NIP. In some embodiments, the polyacrylamides-based copolymer comprises about 20% to about 26% by weight of the functional dopant monomer NIP.

In some embodiments, the polyacrylamides-based copolymer comprises MORPH as the water-soluble carrier monomer and about 2% to about 30% by weight of NIP as the functional dopant monomer. In some embodiments the, copolymer comprises MORPH and from about 5% to about 30% by weight of NIP. In some embodiments, the copolymer comprises MORPH and about 10% to about 28% by weight of NIP. In some embodiment s, the copolymer comprises MORPH and about 5% to about 26% by weight of NIP. In some embodiments, the copolymer comprises MORPH and about 5% to about 10% by weight of NIP. In some embodiments the, copolymer comprises MORPH and about 10% to about 15% by weight of NIP. In some embodiments, the copolymer comprises MORPH and about 15% to about 20% by weight of NIP. In some embodiments, the copolymer comprises MORPH and about 20% to about 25% by weight of NIP. In some embodiment s, the copolymer comprises MORPH and about 25% to about 30% by weight of NIP. In some embodiments, the copolymer comprises MORPH and about 20% to about 28% by weight of NIP. In some embodiments, the copolymer comprises MORPH and about 21% by weight of NIP. In some embodiments, the copolymer comprises MORPH and about 22% by weight of NIP. In some embodiments, the copolymer comprises MORPH and about 23% by weight of NIP. In some embodiments, the copolymer comprises MORPH and about 24% by weight of NIP. In some embodiments, the copolymer comprises MORPH and about 25% by weight of NIP.

In some embodiments, the polyacrylamide-bas edcopolymer comprises AMP, TMA, TEA, or PHE as the functional dopant monomer. In some embodiments, AMP, TMA, TEA, or PHE functional dopant monomer is present at about 2% to about 16% by weight of the copolymer. In some embodiments AMP,, TMA, TEA, or PHE functional dopant monomer is present at about 5% to about 15% by weight of the copolymer. In some embodiments, AMP, TMA, TEA, or PHE functional dopant monomer is present at about 6% to about 14% by weight of the copolymer. In some embodiments AMP,, TMA, TEA, or PHE functional dopant monomer is present at about 12% to about 15% by weight of the copolymer. In some embodiments, AMP, TMA, TEA, or PHE functional dopant monomer is present at about 2% to about 5% by weight of the copolymer. In some embodiments , AMP, TMA, TEA, or PHE functional dopant monomer is present at about 5% to about % by weight of the copolymer.

In some embodiments, the polyacrylamide-bas edcopolymer comprises MORPH as the water-soluble carrier monomer and about 2% to about 16% by weight of PHE as the functional dopant monomer. In some embodiments, the polyacrylamide-based copolymer comprises MORPH as the water-soluble carrier monomer and about 4% to about 16% by weight of PHE as the functional dopant monomer. In some embodiments ,the polyacrylamide-bas edcopolymer comprises MORPH as the water-soluble carrier monomer and about 6% to about 14% by weight of PHE as the functional dopant monomer. In some embodiments, the polyacrylamide-based copolymer comprises MORPH as the water-soluble carrier monomer and about 8% to about 14% by weight of PHE as the functional dopant monomer. In some embodiments, the polyacrylamide-based copolymer comprises MORPH as the water-soluble carrier monomer and about 10% to about 14% by weight of PHE as the functional dopant monomer. In some embodiment s, the polyacrylamide-based copolymer comprises MORPH as the water-soluble carrier monomer and about 10% to about 12% by weight of PHE as the functional dopant monomer.

In some embodiments the, polyacrylamide-bas edcopolymer comprises MP AM as the water-soluble carrier monomer and about 2% to about 16% by weight of PHE as the functional dopant monomer. In some embodiments, the polyacrylamide-based copolymer comprises MP AM as the water-soluble carrier monomer and about 5% to about 15% by weight of PHE as the functional dopant monomer. In some embodiments ,the polyacrylamide-bas copolymered comprises MP AM as the water-soluble carrier monomer and about 6% to about 10% by weight of PHE as the functional dopant monomer. In some embodiments, the polyacrylamide-based copolymer comprises MPAM as the water- solubl carrie er monomer and about 7% by weight of PHE as the functional dopant monomer. In some embodiments, the polyacrylamide-base copolymerd comprises MPAM as the water-soluble carrier monomer and about 8% by weight of PHE as the functional dopant monomer. In some embodiments, the polyacrylamide-based copolymer comprises MPAM as the water-soluble carrier monomer and about 9% by weight of PHE as the functional dopant monomer.

In some embodiments the, polyacrylamide-based copolymer comprises about 3% to about 17% by weight of TRI as the functional dopant monomer. In some embodiment s, the polyacrylamide-bas edcopolymer comprises about 7% to about 12% by weight of TRI as the functional dopant monomer. In some embodiments, the polyacrylamide-based copolymer comprises about 4% to about 6% by weight of TRI as the functional dopant monomer. In some embodiments, the polyacrylamide-based copolymer comprises about 13% to about 17% by weight of TRI as the functional dopant monomer.

In some embodiments, the degree of polymerizatio (DP)n of the polyacrylamide- based copolymer is about 10 to about 500, about 20 to about 200, about 50 to about 100, about 100 to about 200, about 200 to about 300, about 300 to about 400, or about 400 to about 500, or about 10, about 50, about 70, about 100, about 120, about 150, about 170, about 200, about 220, about 250, about 270, about 300, about 320, about 350, about 370, about 400, about 420, about 450, about 470, or about 500. In some embodiments the, DP of the copolymer is about 40. In some embodiments the, DP of the copolymer is about 50.

In some embodiments, the DP of the copolymer is about 60. In some embodiments the, DP of the copolymer is about 70. In some embodiments the, DP of the copolymer is about 80. In some embodiments, the DP of the copolymer is about 90. In some embodiments , the DP of the copolymer is about 100.

In some embodiments, the molecula weir ght of the polyacrylamide-based copolymer is about 1,000 g/mol to about 40,000 g/mol, such as about 1,000 g/mol to about ,000 g/mol, about 1,000 g/mol to about 30,000 g/mol ,about 1,000 g/mol to about 25,000 g/mol ,about 1,000 g/mol to about 20,000 g/mol, about 1,000 g/mol to about 15,000 g/mol, about 1,000 g/mol to about 10,000 g/mol, about 1,000 g/mol to about 7,000 g/mol, about 1,000 g/mol to about 6,000 g/mol, about 1,000 g/mol to about 5,000 g/mol, about 1,000 g/mol to about 4,000 g/mol, about 1,000 g/mol to about 3,000 g/mol, about 3,000 g/mol to about 40,000 g/mol ,about 3,000 g/mol to about 35,000 g/mol ,about 3,000 g/mol to about ,000 g/mol, about 3,000 g/mol to about 25,000 g/mol ,about 3,000 g/mol to about 20,000 g/mol ,about 3,000 g/mol to about 15,000 g/mol, about 3,000 g/mol to about 10,000 g/mol, about 3,000 g/mol to about 7,000 g/mol ,about 3,000 g/mol to about 6,000 g/mol ,about 3,000 g/mol to about 5,000 g/mol ,about 3,000 g/mol to about 4,000 g/mol ,about 4,000 g/mol to about 40,000 g/mol ,about 4,000 g/mol to about 35,000 g/mol ,about 4,000 g/mol to about 30,000 g/mol ,about 4,000 g/mol to about 25,000 g/mol ,about 4,000 g/mol to about 20,000 g/mol ,about 4,000 g/mol to about 15,000 g/mol ,about 4,000 g/mol to about ,000 g/mol, about 4,000 g/mol to about 7,000 g/mol, about 4,000 g/mol to about 6,000 g/mol ,about 4,000 g/mol to about 5,000 g/mol ,about 5,000 g/mol to about 40,000 g/mol, about 5,000 g/mol to about 35,000 g/mol ,about 5,000 g/mol to about 30,000 g/mol ,about ,000 g/mol to about 25,000 g/mol ,about 5,000 g/mol to about 20,000 g/mol ,about 5,000 g/mol to about 15,000 g/mol ,about 5,000 g/mol to about 10,000 g/mol ,about 5,000 g/mol to about 7,000 g/mol ,about 5,000 g/mol to about 6,000 g/mol ,about 6,000 g/mol to about 40,000 g/mol, about 6,000 g/mol to about 35,000 g/mol ,about 6,000 g/mol to about 30,000 g/mol ,about 6,000 g/mol to about 25,000 g/mol, about 6,000 g/mol to about 20,000 g/mol, about 6,000 g/mol to about 15,000 g/mol, about 6,000 g/mol to about 10,000 g/mol ,about 6,000 g/mol to about 7,000 g/mol ,about 7,000 g/mol to about 40,000 g/mol, about 7,000 g/mol to about 35,000 g/mol ,about 7000 g/mol to about 30,000 g/mol ,about 7,000 g/mol to about 25,000 g/mol ,about 7,000 g/mol to about 20,000 g/mol ,about 7,000 g/mol to about 15,000 g/mol, about 7,000 g/mol to about 10,000 g/mol, about 10,000 g/mol to about 40,000 g/mol ,about 10,000 g/mol to about 35,000 g/mol ,about 10,000 g/mol to about 30,000 g/mol, about 10,000 g/mol to about 25,000 g/mol, about 10,000 g/mol to about ,000 g/mol, about 10,000 g/mol to about 15,000 g/mol, about 15,000 g/mol to about 40,000 g/mol ,about 15,000 g/mol to about 35,000 g/mol ,about 15,000 g/mol to about ,000 g/mol ,about 15,000 g/mol to about 25,000 g/mol ,about 15,000 g/mol to about ,000 g/mol ,about 20,000 g/mol to about 40,000 g/mol ,about 20,000 g/mol to about ,000 g/mol ,about 20,000 g/mol to about 30,000 g/mol ,about 20,000 g/mol to about ,000 g/mol ,about 25,000 g/mol to about 40,000 g/mol ,about 25,000 g/mol to about ,000 g/mol ,about 25,000 g/mol to about30,000 g/mol ,about 30,000 g/mol to about 40,000 g/mol, about 30,000 g/mol to about 35,000 g/mol ,or about 35,000 g/mol to about 40,000 g/mol. In some embodiments, the molecul weiar ght of the copolymer is about 1,000 to about 30,000 g/mol. In some embodiments, the molecula weir ght of the copolymer is about 10,000 to about 20,000 g/mol. In some embodiments, the molecula weir ght of the copolymer is about 15,000 to about 20,000 g/mol. In some embodiments the, molecular weight of the copolymer is about 20,000 to about 25,000 g/mol. the molecula weir ght of the copolymer is about 25,000 to about 30,000 g/mol. In some embodiments, the molecula weighr t of the copolymer is about 30,000 to about 40,000 g/mol. In some embodiments, the molecula weir ght of the copolymer is about 2,000 to about 10,000 g/mol. In some embodiments, the molecula weir ght of the copolymer is about 3,000 to about 7,000 g/mol. In some embodiments the, molecula weir ght of the copolymer is about 4,000 to about 6,000 g/mol.

Also provided in the present disclosure is a polyacrylamide-bas edcopolymer that contains a water-soluble carrier monomer comprising an acrylamide reactive moiety and a functional dopant monomer comprising an acrylamide reactive moiety. In some embodiments, the polyacrylamide-bas edcopolymer comprises about 70% to about 98% of a water-soluble carrier monomer wit han acrylamide reactive moiety and about 2% to about 30% of a functional dopant monomer with an acrylamide reactive moiety. In some embodiments, the number-averaged molecula weir ght (Mn) of the copolymer is about 1,000 g/mol to about 30,000 g/mol .In some embodiments, the degree of polymerization is about 10 to about 250. In some embodiments, the water-solubl carrie er monomer is non- ionic. In some embodiments, the functional dopant monomer is hydrophobic. In some embodiments, the copolymer is amphiphilic.

In some embodiments the, water-soluble carrier monomer is selected from the group consisting of N-(3-methoxypropoyl)acrylamide (MP AM), 4-acryloylmorpholine (MORPH), N,N-dimethylacrylam ide(DMA), N-hydroxyethyl acrylamide (HEAM), and acrylamide (AM). In some embodiments, the water-soluble carrier monomer is selected from the group consisting of N-(3-methoxypropoyl)acrylam ide(MPAM) and 4- acryloylmorpholine (MORPH). In some embodiments, the water-soluble carrier monomer is N-(3-methoxypropoyl)acrylamide (MPAM). In some embodiments, the water-solubl e carrier monomer is 4-acryloylmorpholine (MORPH).

In some embodiments, the functional dopant monomer is selected from the group consisting of N-[tris(hydroxymethyl)-methyl]acrylam (TRI)ide , 2-acrylamido-2- methylpropane sulfonic acid (AMP), (3-acrylamidopropyl)trimethylammo chloridenium (TMA), N-isopropylacrylamide (NIP), N-tert-butyl acrylamide (TEA), and N- phenyl acrylamide (PHE). In some embodiments, the functional dopant monomer is selected from the group consisting of N-isopropylacrylamide (NIP) and N- phenyl acrylamide (PHE). In some embodiments, the functional dopant monomer is N- isopropyl acrylamide (NIP). In some embodiments the, functional dopant monomer is N- phenyl acrylamide (PHE).

In some embodiments, the polyacrylamide-base copolymerd comprises about 70% to about 95% by weight of the water-soluble carrier monomer MORPH and about 5% to about 30% by weight of the functional dopant monomer NIP, wherein the number- averaged molecula weir ght (Mn) of the copolymer is about 1,000 g/mol to about 10,000 g/mol and the degree of polymerizatio isn about 10 to about 100. In some embodiments , the polyacrylamide-based copolymer comprises about 74% to about 80% by weight of the water-soluble carrier monomer MORPH and about 20% to about 26% by weight of the functional dopant monomer NIP, wherein the number-averaged molecula weir ght (Mn) of the copolymer is about 1,000 g/mol to about 5,000 g/mol and the degree of polymerization is about 10 to about 50. In some embodiments, the polyacrylamide-based copolymer comprises about 77% by weight of the water-soluble carrier monomer MORPH and about 23% by weight of the functional dopant monomer NIP, wherein the number-averaged molecula weir ght (Mn) of the copolymer is about 3,200 g/mol and the degree of polymerization is about 26.

Compositions containing a polyacrylamide-based copolymer Also provided are compositions containing the polyacrylamide-bas edcopolymers described in the present disclosure. In some embodiments the, composition comprises a polyacrylamide-bas edcopolymer of the present disclosure and a pharmaceutical ly acceptable excipient.

In some embodiments, the composition comprises a polyacrylamide-based copolymer of the present disclosure and a biologic molecule. In some embodiments the, biologic molecule is a protein. In some embodiments the, protein is a protein susceptibl e to aggregation in an aqueous medium. In some embodiments the, copolymer is uncharged, cationic, or anionic. In some embodiments the, copolymer is amphiphilic. In some embodiments, the carrier monomers are the water-solubl specie es and are responsible for both maintaining solubilit andy providing an inert barrier to prevent aggregation of the biological species , for example, to prevent protein-protein interactions. In some embodiments, the water-soluble carrier monomer is the predominant species within the polyacrylamide-bas copolymer.ed In some embodiments the, functional dopant monomers are copolymerized at lower weight percentages and are incorporated statistica lly throughout the resulting copolymer. In some embodiments these, dopants are selected to promote either polymer-interface interactions or polymer-protein interactions.

In some embodiments the, composition that contains a polyacrylamide-based copolymer of the present disclosure and a biologic molecule or lipid-based vehicl ehas a copolymer concentration of about 0.0001% to about 5% by weight of the composition, such as about 0.0001% to about 4%, about 0.0001% to about 3%, about 0.0001% to about 2%, about 0.0001% to about 1%, about 0.0001% to about 0.5%, about 0.0001% to about 0.4%, about 0.0001% to about 0.3%, about 0.0001% to about 0.2%, about 0.0001% to about 0.1%, about 0.0001% to about 0.05%, about 0.0001% to about 0.02%, about 0.0001% to about 0.01%, about 0.0001% to about 0.005%, about 0.005% to about 5%, about 0.005% to about 4%, about 0.005% to about 3%, about 0.005% to about 2%, about 0.005% to about 1%, about 0.005% to about 0.5%, about 0.005% to about 0.4%, about 0.005% to about 0.3%, about 0.005% to about 0.2%, about 0.005% to about 0.1%, about 0.005% to about 0.05%, about 0.005% to about 0.02%, about 0.005% to about 0.01%, about 0.01% to about 5%, about 0.01% to about 4%, about 0.01% to about 3%, about 0.01% to about 2%, about 0.01% to about 1%, about 0.01% to about 0.5%, about 0.01% to about 0.4%, about 0.01% to about 0.3%, about 0.01% to about 0.2%, about 0.01% to about 0.1%, about 0.01% to about 0.05%, about 0.01% to about 0.02%, about 0.02% to about 5%, about 0.02% to about 4%, about 0.02% to about 3%, about 0.02% to about 2%, about 0.02% to about 1%, about 0.02% to about 0.5%, about 0.02% to about 0.4%, about 0.02% to about 0.3%, about 0.02% to about 0.2%, about 0.02% to about 0.1%, about 0.02% to about 0.05%, about 0.05% to about 5%, about 0.05% to about 4%, about 0.05% to about 3%, about 0.05% to about 2%, about 0.05% to about 1%, about 0.05% to about 0.5%, about 0.05% to about 0.4%, about 0.05% to about 0.3%, about 0.05% to about 0.2%, about 0.05% to about 0.1%, about 0.1% to about 5%, about 0.1% to about 4%, about 0.1% to about 3%, about 0.1% to about 2%, about 0.1% to about 1%, about 0.1% to about 0.5%, about 0.1% to about 0.4%, about 0.1% to about 0.3%, about 0.1% to about 0.2%, about 0.2% to about 5%, about 0.2% to about 4%, about 0.2% to about 3%, about 0.2% to about 2%, about 0.12% to about 1%, about 0.2% to about 0.5%, about 0.2% to about 0.4%, about 0.2% to about 0.3%, about 0.3% to about 5%, about 0.3% to about 4%, about 0.3% to about 3%, about 0.3% to about 2%, about 0.3% to about 1%, about 0.3% to about 0.5%, about 0.3% to about 0.4%, about 0.4% to about 5%, about 0.4% to about 4%, about 0.4% to about 3%, about 0.4% to about 2%, about 0.4% to about 1%, about 0.4% to about 0.5%, about 0.5% to about 5%, about 0.5% to about 4%, about 0.5% to about 3%, about 0.5% to about 2%, about 0.5% to about 1%, about 1% to about 5%, about 1% to about 4%, about 1% to about 3%, about 1% to about 2%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, about 3% to about 5%, about 3% to about 4%, or about 4% to about % by weight of the composition. In some embodiments, the copolymer concentration is about 0.0001% to about 5% by weight of the composition. In some embodiments, the copolymer concentration is about 0.001% to about 1% by weight, about 0.005% to about 0.5% by weight, about 0.005% to about 0.02% by weight, about 0.01% to about 0.2% by weight, about 0.1% to about 0.4% by weight, or about 0.2% to about 0.3% by weight of the composition. In some embodiments the, copolymer concentration is about 0.005% by weight of the composition. In some embodiments, the copolymer concentration is about 0.01% by weight of the composition. In some embodiments, the copolymer concentration is about 0.05% by weight of the composition. In some embodiments, the copolymer concentration is about 0.1% by weight of the composition. In some embodiments, the copolymer concentration is about 1% by weight of the composition.

In some embodiments the, protein is selected from the group consisting of antibodies and fragment sthereof, cytokines ,chemokines, hormones, vaccine antigens, cancer antigens, adjuvants and, combinations thereof. In some embodiments the, protein is an antibody. In some embodiments, the protein is a monoclonal antibody. In some embodiments, the protein is a vaccine. In some embodiments, the protein is a hormone. In some embodiments, the composition exhibits reduced aggregation of the protein as compared to a composition of the same protein without the copolymer. In some embodiments, the composition exhibits reduced precipitation of the protein as compared to a composition of the same protein without the copolymer. In some embodiments the, composition comprises the protein in a concentration at least two times greater, at leas t three time sgreater, at least four times greater, or at least five times greater than the concentration of the same protein in the composition without the copolymer.

In some embodiments, the protein is insulin, or an analog thereof. Thus, also provided are compositions that contain a polyacrylamide-based copolymer of the present disclosure and insulin, or an analog thereof.

Non-limiting example sof insulin and insuli nanalogs include insulin lispro, HUMALOG® (fast-acting insulin lispro), insulin glargine, LANTUS® (insulin glargine), insulin detemir, LEVEMIR® (insuli ndetemir ),ACTRAPID® (fast-acting human insulin), modern insulin, NOVORAPID® (insulin aspart), VELOSULIN® (human insulin), HUMULIN® M3 (a mixture of solubl insule in and isophane insulin called biphasic isophane insulin), HYPURIN®® (neutral bovine insulin), INSUMAN® (recombinant human insulin), INSULATARD® (long-acting isophane human insulin), MIXTARD® 30 (a mixture of 30% solubl insulie nand 70% isophane insulin), MIXTARD® 40 (a mixture of 40% solubl insule in and 60% isophane insulin), MIXTARD® 50 (a mixture of 50% solubl e insulin and 50% isophane insulin ),insulin aspart, insulin glulisine, insulin isophane, insulin degludec insul, in icodec, insuli nzinc extended, NOVOLIN® R (human insulin), HUMULIN® R (human insulin), HUMULIN® R regular U-500 (concentrated regular insulin), NOVOLIN* N (intermediate-acting human insulin), HUMULIN® N (intermediate-acti nghuman insulin), RELION® (over-the-counter brand of NOVOLIN® R, NO VOLIN® N, and NO VOLIN® 70/30), AFREZZA® (rapid-acting inhaled insulin), HUMULIN® 70/30 (a mixture of 70% human insulin isophane suspension and 30% human insulin injection), NOVOLIN® 70/30 (a mixture of 70% NPH, human insulin isophane suspension and 30% regular, human insulin injection), NOVOLOG® 70/30 (a mixture of 70% insulin aspart protamine suspension and 30% insuli naspart injection), HUMULIN® 50/50 (a mixture of 50% human insuli nisophane suspension and 50% human insulin injection), HUMALOG® Mix 75/25 (a mixture of 75% insulin lispro protamine suspension and 25% insulin lispro injection), insulin aspart protamine-insul aspartin ,insulin lispro protamine-insul inlispro, insulin lispro protamine-insul inlispro, human insulin NPH- human insulin regular, insuli ndegludec-insul aspain rt, and combinations thereof. In some embodiments, the insulin, or an analog thereof, is a human insuli nor a recombinant human insulin. In some embodiments the, insulin, or an analog thereof, is a non-human (e.g., primate, pig, cow, cat, dog, or rodent insuli) orn a recombinant non-human insulin. In some embodiments, the insulin, or an analog thereof, is a purified or synthet icinsulin. In some embodiments, the insulin, or an analog thereof, is selected from the group consisting of a rapid-acting insulin, a short-acting insulin, an intermediate-act inginsulin, a long-acting insulin, and a pre-mixed insulin. In some embodiments the, insulin, or an analog thereof, is insulin lispro. In some embodiments the, insulin, or an analog thereof, is HUMALOG®, a commerciall yavailabl efast-acting human insulin analog, insuli nlispro. In some embodiments, the insulin, or an analog thereof, is insulin aspart . In some embodiment s, the insulin, or an analog thereof, is insuli nglulisine. In some embodiments the, insulin, or an analog thereof, is recombinant human insulin.

In some embodiments, the insulin, or an analog thereof, is present in the composition in the monomeric form, dimeric form, hexameric form, and combinations thereof. In some embodiments about, 1 mol% to about 5 mol% ,about 1 mol% to about mol%, about 1 mol % to about 20 mol% ,about 1 mol % to about 30 mol% ,about 1 mol% to about 40 mol% ,about 1 mol% to about 50 mol% ,about 1 mol% to about 60 mol%, about 1 mol% to about 70 mol% , about 1 mol% to about 80 mol%, about 1 mol% to about 90 mol%, or about 10 mol % to about 100 mol % of the insulin, or analog thereof, is present in the composition in monomeric form. For example, about 1 mol% ,about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 15 mol%, about 20 mol%, about 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol% ,about 60 mol% ,about 65 mol% ,about 70 mol% ,about 75 mol%, about 80 mol%, about 85 mol%, about 90 mol%, about 95 mol%, about 99 mol%, or about 100 mol% of the insulin, or an analog thereof, is present in the composition in monomeric form. In some embodiments, about 3 mol% of the insulin, or an analog thereof, is present in the composition in monomeric form. In some embodiments insulin,, or an analog thereof, is "substantia llypresent" in a composition in the monomeric form, which means greater than about 50 mol% of the insulin, or insulin analog, is present in monomeric form. In some embodiments, the insulin, or an analog thereof, comprises about 50 mol% or greater insulin in monomeric form. In some embodiments, the insulin, or an analog thereof, comprises about 60 mol% or greater insulin in monomeric form. In some embodiments, the insulin, or an analog thereof, comprises about 70 mol% or greater insuli nin monomeric form. In some embodiments, the insulin, or an analog thereof, comprises about 80 mol% or greater insulin in monomeric form. In some embodiments the, insulin, or an analog thereof, comprises about 90 mol% or greater insulin in monomeric form. In some embodiment s, the insulin, or an analog thereof, comprises about 99 mol% or greater insulin in monomeric form. In some embodiments, the insulin, or an analog thereof, comprises about 100 mol% insulin in monomeric form. In some embodiments, insulin, or an analog thereof, is "substantia llyabsent" in a composition in the monomeric form, which means les sthan about 10 mol% of the insulin, or insulin analog thereof, is present in monomeric form.

In some embodiments, about 10 mol % to about 100 mol % of the insulin, or analog thereof, is present in the composition in dimeric form. In some embodiments, about 10 mol % to about 100 mol% ,about 20 mol % to about 90 mol%, about 30 mol % to about 80 mol% ,about 40 mol% to about 70 mol%, about 50 mol% to about 60 mol%, about 60 mol % to about 100 mol% ,about 80 mol % to about 100 mol% ,about 90 mol% to about 100 mol%, or about 95 mol% to about 100 mol% of the insulin, or analog thereof, is present in the composition in dimeric form. For example, about 10 mol% ,about 15 mol%, about 20 mol%, about 25 mol%, about 30 mol%, about 35 mol%, about 40 mol% ,about 45 mol% ,about 50 mol% ,about 55 mol% ,about 60 mol%, about 65 mol%, about 70 mol%, about 75 mol%, about 80 mol% ,about 85 mol% ,about 90 mol%, about 95 mol%, about 97 mol%, about 99 mol%, or about 100 mol % of the insulin, or an analog thereof, is present in the composition in dimeric form. In some embodiments, insulin, or an analog thereof, is "substantiall presenty " in a composition in the dimeric form, which means greater than about 50 mol% of the insulin, or insuli nanalog, is present in dimeric form. In some embodiments, the insulin, or an analog thereof, comprises about 50 mol% or greater insulin in dimeric form. In some embodiments, the insulin, or an analog thereof, comprises about 60 mol% or greater insulin in dimeric form. In some embodiments the, insulin, or an analog thereof, comprises about 70 mol% or greater insulin in dimeric form. In some embodiments, the insulin, or an analog thereof, comprises about 80 mol% or greater insulin in dimeric form. In some embodiments, the insulin, or an analog thereof, comprises about 90 mol% or greater insulin in dimeric form. In some embodiments, the insulin, or an analog thereof, comprises about 97 mol% or greater insulin in dimeric form. In some embodiments, the insulin, or an analog thereof, comprises about 99 mol% or greater insulin in dimeric form. In some embodiments, the insulin, or an analog thereof, comprises about 100 mol% insulin in dimeric form.

In some embodiments, the insulin is not conjugated to poly(ethylene glycol (PEG)) or a trehalose polymer.

The concentration range of insulin, or analog thereof, can be from about 0.34 mg/mL (10 U/mL) to about 34 mg/mL (1000 U/mL). In some embodiments, the insulin concentration is about 1.7 mg/mL (50 U/mL) to about 17 mg/mL (500 U/mL). In some embodiments, the insulin concentration is about 17 mg/mL (500 U/mL) to about 34 mg/mL (1000 U/mL). In some embodiments the, insulin concentration is about 3.4 mg/mL (100 U/mL). In some embodiments the, insulin concentration is about 6.8 mg/mL (200 U/mL).

In some embodiments the, insulin concentration is about 10.2 mg/mL (300 U/mL). In some embodiments, the insulin concentration is about 13.6 mg/mL (400 U/mL). In some embodiments, the insulin concentration is about 17 mg/mL (500 U/mL). In some embodiments, the insulin concentration is about 20.4 mg/mL (600 U/mL). In some embodiments, the insulin concentration is about 23.8 mg/mL (700 U/mL). In some embodiments, the insulin concentration is about 27.2 mg/mL (800 U/mL). In some embodiments, the insulin concentration is about 30.6 mg/mL (900 U/mL). In some embodiments, the insulin concentration is about 34 mg/mL (1000 U/mL).

Thus, also provided are compositions comprising a polyacrylamide-based copolymer of the present disclosure and insulin. In some embodiments ,the polyacrylamide-bas edcopolymer contains a MORPH carrier monomer and a NIP dopant monomer. In some embodiments, the composition contains about 0.005 wt% to about 0.2 wt% of a polyacrylamide-base copolymd er comprising about 70% to about 95% by weight of a MORPH carrier monomer and about 5% to about 30% by weight of a NIP dopant monomer; and about 100 U/mL insulin, or an analog thereof. In some embodiments the, composition contains about 0.01 wt% of a polyacrylamide-based copolymer comprising about 74% to about 80% by weight of a MORPH carrier monomer and about 20% to about 26% by weight of a NIP dopant monomer; and about 100 U/mL insulin, or an analog thereof. In some embodiments, the composition contains about 0.01 wt% of a polyacrylamide-bas edcopolymer comprising about 77% by weight of a MORPH carrier monomer and about 23% by weight of a NIP dopant monomer; and about 100 U/mL insulin, or an analog thereof.

In some embodiments, the copolymers described in the present disclosure reduce or prevent insulin aggregation. In some embodiments, the copolymers enable the stable formulati onof insulin, or an analog thereof, in its monomeric form. In some embodiment s, about 10 mol % to about 100 mol % of the insulin molecules, or analog thereof, in the formulati onare present as monomeric insulin, or an analog thereof. For example, about 10 mol%, about 15 mol%, about 20 mol% ,about 25 mol% ,about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, about 60 mol% ,about 65 mol% ,about 70 mol% ,about 75 mol% ,about 80 mol%, about 85 mol%, about 90 mol%, about 95 mol%, about 99 mol% ,or about 100 mol % of the insulin molecule ors, an analog thereof, in the formulation are present as monomeric insulin. In some embodiment s, more than about 50 mol% of the insulin molecules, or an analog thereof, in the formulati on are present as monomeric insulin, or an analog thereof. In some embodiments, more than about 60 mol% of the insulin molecule s,or an analog thereof, in the formulati onare present as monomeric insulin, or an analog thereof. In some embodiments, more than about 70 mol% of the insulin molecules, or an analog thereof, in the formulati onare present as monomeric insulin, or an analog thereof. In some embodiments more, than about 80 mol% of the insulin molecules, or an analog thereof, in the formulati onare present as monomeric insulin, or an analog thereof. In some embodiments, more than about 90 mol% of the insulin molecule s,or an analog thereof, in the formulati onare present as monomeric insulin, or an analog thereof. In some embodiments, about 100 mol % of the insulin molecules, or an analog thereof, in the formulati onare present as monomeric insulin, or an analog thereof. In some embodiments the, formulati oncontains up to about 100 mol % insulin, or an analog thereof, present as dimeric insulin, or an analog thereof. In some embodiments, les sthan about 90 mol% of the insulin molecules, or an analog thereof, in the formulati onare present as dimeric insulin, or an analog thereof. In some embodiment s, les sthan about 80 mol% of the insuli nmolecules, or an analog thereof, in the formulati on are present as dimeric insulin, or an analog thereof. In some embodiments, less than about 70 mol% of the insulin molecules, or an analog thereof, in the formulati onare present as dimeric insulin, or an analog thereof. In some embodiments, less than about 60 mol% of the insulin molecules, or an analog thereof, in the formulati onare present as dimeric insulin, or an analog thereof. In some embodiments, les sthan about 50 mol% of the insuli n molecules, or an analog thereof, in the formulati onare present as dimeric insulin, or an analog thereof. In some embodiments, les sthan about 40 mol% of the insulin molecules , or an analog thereof, in the formulati onare present as dimeric insulin, or an analog thereof.

In some embodiments les, sthan about 30 mol % of the insulin molecules, or an analog thereof, in the formulati onare present as dimeric insulin, or an analog thereof. In some embodiments, les sthan about 20 mol% of the insulin molecules, or an analog thereof, in the formulati onare present as dimeric insulin, or an analog thereof. In some embodiment s, les sthan about 10 mol % of the insuli nmolecules, or an analog thereof, in the formulati on are present as dimeric insulin, or an analog thereof. In some embodiments, the formulati on is essentiall freey of dimeric insulin, or an analog thereof. In some embodiments about, 0 mol % to about 100 mol % of the insulin molecule ors, an analog thereof, in the formulati on are present as hexameric insulin. In some embodiments, more than about 95 mol% of the insuli molen cule ors, an analog thereof, in the formulati onare present as hexameric insulin, or an analog thereof. In some embodiments les, sthan about 99 mol% of the insuli n molecules, or an analog thereof, in the formulation are present as hexameric insulin, or an analog thereof. In some embodiments, les sthan about 90 mol% of the insulin molecules , or an analog thereof, in the formulation are present as hexameric insulin, or an analog thereof. In some embodiments, less than about 80 mol% of the insulin molecule s,or an analog thereof, in the formulati onare present as hexameric insulin, or an analog thereof.

In some embodiments les, sthan about 70 mol% of the insulin molecules, or an analog thereof, in the formulati onare present as hexameric insulin, or an analog thereof. In some embodiments, les sthan about 60 mol% of the insulin molecules, or an analog thereof, in the formulati onare present as hexameric insulin, or an analog thereof. In some embodiments, les sthan about 50 mol% of the insulin molecules, or an analog thereof, in the formulati onare present as hexameric insulin, or an analog thereof. In some embodiments, les sthan about 30 mol % of the insulin molecules, or an analog thereof, in the formulati onare present as hexameric insulin, or an analog thereof. In some embodiments, les sthan about 20 mol% of the insulin molecules, or an analog thereof, in the formulati onare present as hexameric insulin, or an analog thereof. In some embodiments, les sthan about 10 mol % of the insulin molecules, or an analog thereof, in the formulati onare present as hexameric insulin, or an analog thereof. In some embodiments, les sthan about 5 mol% of the insulin molecule s,or an analog thereof, in the formulati onare present as hexameric insulin, or an analog thereof. In some embodiments, the formulati onis essentiall freey of hexameric insulin, or an analog thereof.

In some embodiments about, 0 mol% of the insulin molecules, or an analog thereof, in the formulati onare present as hexameric insulin, or an analog thereof. In some embodiments , the formulati ondoes not comprise Zn(II).

In some embodiments, the stabl eformulati onof insuli nin its monomeric form results in ultra-fast kinetics in vivo. In some embodiments the, copolymers enable the preparation of an ultrafast-absorbing insulin lispro (UFAL). In some embodiments more, than 50 mol% of the insuli nmolecules in the UFAL formulati onare present as monomeric insulin. In some embodiments, more than 60 mol% of the insulin molecules in the UFAL formulati onare present as monomeric insulin. In some embodiments, more than 70 mol% of the insulin molecule ins the UFAL formulati onare present as monomeric insulin. In some embodiments, more than 80 mol% of the insulin molecules in the UFAL formulati on are present as monomeric insulin. In some embodiments more, than 90 mol% of the insuli n molecules in the UFAL formulati onare present as monomeric insulin. In some embodiments, les sthan 30 mol% of the insulin molecules in the UFAL formulati onare present as dimeric insulin. In some embodiments, less than 20 mol% of the insuli n molecules in the UFAL formulation are present as dimeric insulin. In some embodiment s, les sthan 10 mol % of the insuli moleculn esin the UFAL formulati onare present as dimeric insulin. In some embodiments the, UFAL formulati onis essentially free of dimeric insulin.

In some embodiments, less than 30 mol% of the insulin molecule ins the UFAL formulati onare present as hexameric insulin. In some embodiments, less than 20 mol% of the insuli nmolecule ins the UFAL formulati onare present as hexameric insulin. In some embodiments, les sthan 10 mol% of the insulin molecules in the UFAL formulati onare present as hexameric insulin. In some embodiments less, than 5 mol% of the insulin molecules in the UFAL formulati onare present as hexameric insulin. In some embodiments, the UFAL formulati onis essentiall freey of hexameric insulin.

In some embodiments, the composition comprises insulin, or an analog thereof, substantiall presenty in monomeric form, where administration of the composition to a subject results in a shorter duration of action as compared to administration of a composition comprising the same amount of insulin, or an analog thereof, substantiall y present in dimeric form, hexameric form, or a combination thereof, where the duration of action is the time to depletion of 50% of the maximum concentration observed (Umax).

In some embodiments, the composition comprises insulin, or an analog thereof, substantiall presenty in monomeric form, where administration of the composition to a subject results in a shorter time to insuli nonset as compared to administration of a composition comprising the same amount of insulin, or an analog thereof, substantiall y present in dimeric form, hexameric form, or a combination thereof, where the time to onset is the time to 50% of the maximum concentration observed (Umax).

In some embodiments, the composition comprises insulin, or an analog thereof, substantiall presenty in monomeric form, where administration of the composition to a subject resul tsin a greater fraction of total exposure to insulin as compared to administrati onof a composition comprising the same amount of insulin, or an analog thereof, substantia llypresent in dimeric form, hexameric form, or a combination thereof, where the exposure is the fraction of the area under the curve (AUG) at a given timepoint over the total AUG (AUCtimc/AUCtotal).

In some embodiments, the composition comprises insulin, or an analog thereof, substantiall presenty in monomeric form, where administration of the composition to a subject resul tsin a shorter time to maximum concentration of insulin observed (Tmax) as compared to administration of a composition comprising the same amount of insulin, or an analog thereof, substantia llypresent in dimeric form, hexameric form, or a combination thereof.

In some embodiments, the composition comprising a copolymer of the present disclosure and a biologic molecule furthe rcomprise one or more of a pharmaceutical ly acceptable carrier, an aqueous buffer, a tonicity modifier, and a preservative. As used herein, pharmaceuticall acceptabley carriers, tonicity modifiers ,and preservatives are nontoxic to recipients at the dosages and concentrations employed. In some embodiments , the composition contains a buffer, such as phosphate, citrate ,succinat e,other organic acids, and histidine wher, e the term "buffer" refers to a mixture of a weak acid and its conjugat ebase, or vice versa, that is used to maintain the pH of a solution at a nearly constant value. In some embodiments the, buffer comprises one or more phosphate salt s.

In some embodiments, the buffer is sodium phosphate.

In some embodiments the, composition contains a tonicity modifier, such as sodium chloride, potassium chloride mannit, ol, dextros e,glycerol, or magnesium chloride.

In some embodiments the, tonicity modifier is sodium chloride (NaCl )or glycerol In. some embodiments, the tonicity modifier is glycerol.

In some embodiments the, composition contains one or more preservatives, such as phenoxyethanol, phenol , meta-cresol, methylparaben, propylparaben, and benzyl alcohol. In some embodiments, the one or more preservative compris se phenoxyethanol and phenol. In some embodiments, the one or more preservatives comprise phenoxyethanol and meta-cresol. In some embodiments ,the preservative is phenoxyethanol. In some embodiments the, preservative is phenol or meta-cresol.

In some embodiments, the composition comprising the copolymers of the present disclosure is an aqueous composition. In some embodiments, the composition comprising the copolymers of the present disclosure comprises essentiall watery .

In some embodiments, the pH of the composition is about 4 to about 9, such as about 4 to about 8, about 4 to about 7, about 4 to about 6, about 4 to about 5, about 5 to about 9, about 5 to about 8, about 5 to about 7, about 5 to about 6, about 6 to about 9, about 6 to about 8, about 6 to about 7, about 7 to about 9, about 7 to about 8, about 8 to about 9, or about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.4, about 8, about 8.5, or about 9. In some embodiments, the pH of the composition is about 4 to about 9. In some embodiments, the pH of the composition is about 6 to about 8. In some embodiments, the pH of the composition is about 7 to about 8. In some embodiments, the pH of the composition is about 7.4.

In some embodiments the, UFAL comprising the copolymer MORPH-NIP23% is stabl toe stressed aging for over 24 hours. In some embodiments, the UFAL comprising the copolymer MORPH-NIP23% is stabl ealmost four-fold longer than commercial HUMALOG®.

In some embodiments, the UFAL formulations comprising a copolymer of the present disclosure furthe rcomprises one or more of pharmaceuticall acceptabley carriers, tonicity modifiers, and preservatives In. some embodiments, the UFAL formulati on comprises a buffer, a preservative, a tonicity agent, and combinations thereof. In some embodiments, the buffer comprises one or more phosphate salt s.In some embodiments , the buffer is sodium phosphate. In some embodiments, the preservative is phenoxyethanol.

In some embodiments, the preservative is phenol or meta-cresol In. some embodiment s, the tonicity agent is sodium chloride (NaCl) or glycerol In. some embodiments, the tonicit y agent is glycerol.

Also provided are compositions containing the polyacrylamide-bas edcopolymers of the present disclosure and a lipid-based vehicle. In some embodiments, the lipid-based vehiclei sselected from the group consisting of liposomes, polymerosomes mice, lles and, lipid nanoparticles.

In some embodiments the, copolymer sof the present disclosure are used as formulati onadditives in formulations comprising monoclonal antibodies .In some embodiments, the copolymers of the present disclosure are used as formulati onadditives in formulations comprising antibody-drug conjugates. In some embodiments, the copolymer sof the present disclosure are used as formulati onadditives in formulations comprising protein-based vaccine antigens. In some embodiments the, copolymer sof the present disclosure are used as formulati onadditives in formulations comprising protein- polysaccharide conjugate vaccine antigens. In some embodiments, the copolymers of the present disclosure are used as formulati onadditives in formulations comprising nucleic acids. In some embodiments, the copolymers of the present disclosure are used as formulati onadditives in formulations comprising messenger RNA (mRNA). In some embodiments, the copolymers of the present disclosure are used as formulati onadditives in formulations comprising deoxyribose nuclei cacids (DNA). In some embodiments, the copolymer sof the present disclosure are used as formulati onadditives in formulations comprising small-interfering RNA (siRNA). In some embodiments, the copolymers of the present disclosure are used as formulati onadditives in formulations comprising short hairpin RNA (shRNA). In some embodiments, the copolymers of the present disclosure are used as formulati onadditives in formulations comprising microRNA (miRNA). In some embodiments, the copolymers of the present disclosure are used as formulati on additives in formulations comprising one or more nuclei cacids and one or more lipids. In some embodiments, the copolymers of the present disclosure are used as formulati on additives in formulations comprising one or more neutra llipids. In some embodiment s, the copolymers of the present disclosure are used as formulati onadditives in formulations comprising one or more cationic lipids In. some embodiments, the copolymers of the present disclosure are used as formulati onadditives in formulations comprising one or more anionic lipids. In some embodiments the, copolymer sof the present disclosure are used as formulati onadditives in formulations comprising liposomes .In some embodiments, the copolymers of the present disclosure are used as formulati onadditives in formulations comprising lipid nanoparticles. In some embodiments the, copolymers of the present disclosure are used as formulati onadditives in formulations comprising micelles In. some embodiments, the copolymers of the present disclosure are used as formulati onadditives in formulations comprising polymerosomes In. some embodiment s, the copolymers of the present disclosure are used as formulati onadditives in formulations comprising exosomes.

Co-formulations containing insulin Also provided in the present disclosure is a composition containing a polyacrylamide-bas edcopolymer of the present disclosure, insulin, or an analog thereof, and one or more of a peptide, protein, or hormone.

In some embodiments, the co-formulation contains insulin, or an analog thereof, and glucagon, a glucagon-like peptide-1 (GLP-1) receptor agonist, a glucose-dependent insulinotropic polypeptide (GIP) receptor agonist, or a dual GIP and GLP-1 receptor agonist .In some embodiments the, co-formulation comprising a copolymer of the present disclosure and insuli ncomprises a GLP-1 receptor agonist. In some embodiments, the GLP-1 receptor agonist is selected from the group consisting of lixisenatide, liraglutide, albigluti de,dulaglutid exenae, tide, extended-release exenatide, and semaglutide. In some embodiments, the co-formulation comprising a copolymer of the present disclosure and insulin comprises a GLP-1 receptor agonist and a GIP receptor agonist. In some embodiments, the co-formulation comprising a copolymer of the present disclosure and insulin comprises a dual GIP and GLP-1 receptor agonist. In some embodiments, the dual GIP and GLP-1 receptor agonist is tirzepatide. In some embodiments the, co-formulati on comprising a copolymer of the present disclosure and insulin comprises a GIP receptor agonist .In some embodiments the, co-formulation comprising a copolymer of the present disclosure and insulin comprises glucagon.

In some embodiments the, co-formulation comprising a copolymer of the present disclosure and insulin further comprises amylin, or an analog thereof. In some embodiments, the amylin analog is pramlintid e.According to embodiments of the disclosure, co-formulation of monomeric insulin, or an analog thereof, and pramlintide in the presence of a copolymer described herein has ultrafas kinetit cs wit ha high degree of overlap resulting in improved glucos emanagement after a glucose challenge. In some embodiments, co-formulation of monomeric insulin lispro and pramlintide in the presence of a copolymer described herein is shown to have ultrafas kinett ics wit ha high degree of overlap resulting in improved glucose management after a glucos challe enge. As described in Exampl e 3, a formulati oncontaining the amphiphili ccopolymer M0Ni23% as a stabilizing agent is physically stabl etwice as long as commercial HUMALOG® in a stresse agingd assay.

In some embodiments, the composition containing a polyacrylamide-based copolymer of the present disclosure, insulin, or an analog thereof, and amylin, or an amylin analog thereof, are present in a ratio of amylin, or an analog thereof to insulin, or an analog thereof of about 1:1 to about 1:20, about 1:1 to about 1:15, about 1:1 to about 1:10, about 1:1 to about 1:6, or about 1:20, about 1:15, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, or about 1:1.

The copolymers of the present disclosure also enable the formulati onof pramlintide at near neutra lpH (~ pH 7), whereas existing formulations of pramlintide require the pH of the formulation to be at ~ pH 4, which is not compatible wit hinsulin. In some embodiments, the pH of the co-formulati oncomprising a copolymer of the present disclosure, insulin, or an analog thereof, and amylin, or an analog thereof, is at near neutral pH (~ pH 7). In some embodiments, the pH of the co-formulation comprising a copolymer of the present disclosure insul, in, or an analog thereof, and amylin, or an analog thereof, is about 6 to about 8. In some embodiments the, pH of the co-formulation comprising a copolymer of the present disclosure insul, in, or an analog thereof, and amylin, or an analog thereof, is about 6.5. In some embodiments, the pH of the co-formulation comprising a copolymer of the present disclosure insul, in, or an analog thereof, and amylin, or an analog thereof, is about 7. In some embodiments, the pH of the co-formulation comprising a copolymer of the present disclosure insul, in, or an analog thereof, and amylin, or an analog thereof, is about 7.4. In some embodiments the, amylin analog is pramlintide.

Thus, also provided are co-formulation comprising a polyacrylamide-based copolymer of the present disclosure, insulin, or an analog thereof, and pramlintide. In some embodiments, the composition comprises about 0.005 wt% to about 0.2 wt% of a polyacrylamide-bas edcopolymer which comprises about 70% to about 95% by weight of a MORPH carrier monomer and about 5% to about 30% by weight of a NIP dopant monomer, about 100 U/mL insulin, or an analog thereof and about 0.01 mg/mL to about 0.1 mg/mL pramlintide. In some embodiments, the composition comprises about 0.01 wt% of a polyacrylamide-bas copolymed er which comprises about 74% to about 80% by weight of a MORPH carrier monomer and about 20% to about 26% by weight of a NIP dopant monomer, about 100 U/mL insulin, or an analog thereof and about 0.5 mg/mL to about 0.6 mg/mL pramlintide. In some embodiments, the composition comprises about 0.01 wt% of a polyacrylamide-base copolymd er which comprises about 77% by weight of a MORPH carrier monomer and about 23% by weight of a NIP dopant monomer, about 100 U/mL insulin, or an analog thereof and about 0.6 mg/mL pramlintide. In some embodiments the, insulin, or an analog thereof, is substantia llypresent in monomeric form. In some embodiments, the composition does not comprise zinc(II).

In some embodiments, administrati onof the composition to a subject exhibits an insulin AUC6/AUC120 that is at least about 25% greater than the insulin AUC6/AUC120 following administration of HUMALOG® alone or administration of HUMALOG® and pramlintide separately, such as about 30%, 40%, 50%, or greater.

The embodiments of the present disclosure demonstrat thate the copolymer present in the co-formulation does not alter the pharmacokinetic (PK) or pharmacodynami c(PD) properties of the active ingredients within the formulatio n.In some embodiments, the pramlintide in the co-formulation results in delayed gastric emptying similar to separately administered pramlintide. In other embodiments the, combined effects of ultrafas insult in and pramlintide delivery synchronized in the co-formulation of the present disclosure results in reduced glucose depletion below baseline measurements while, maintaining contro lof the initial glucose spike in our simulated "mealtim" eglucos echallenge. Thus , in some embodiments, the co-formulation has potenti alto improve glucos emanagement by reducing the risk of post-prandial hypoglycemia, while reducing patient burden.

In some embodiments use, of the co-formulation results in improved bolus insuli n delivery. In some embodiments ,insulin wit h the ultrafas kinetit cs is delivered synchronously wit hpramlintide in insulin infusion pumps and "artificial pancreas" closed- loop system s.In some embodiments, a stabl insule in-pramlintide co-formulation enables the implementation of dual-hormone treatment in closed-loop systems outside of clinical trials where using two separate infusion pumps is impractical In. some embodiments, the synchronized insulin-pramlintide kinetics and shorte rduration of insulin action in the co- formulati onof the present results in improved autonomous insulin delivery. Typically, closed-loop systems require patients to input carbohydrates counts at mealtimes and are not fully autonomous, in part because insulin absorption kinetics are not rapid enough to reduce mealtim glucosee excursions ,and the extende dduration of insulin action can result in post-prandial hypoglycemia. Thus, an ultrafast insulin-pramlintide co-formulation can rapidly react to mealtim espikes, as the insulin will have immediate onset and the pramlintide will slow the appearance of glucose (through delayed gastri cemptying).

Further, wit hshorte rduration of insuli naction, the risk of hypoglycemia, as a resul oft insulin stacking woul bed reduced.

The present disclosure demonstrate thats a stabl esingle administrati oninsulin- pramlintide co-formulation, utilizing monomeric insulin, can have synchronized ultrafast insulin-pramlintide pharmacokinetic sthat resul int better glycemic contro lin a mealtime simulation. This co-formulation has potential to improve glucos managemente and reduce patient burden in clinical applications using it for both direct bolus administrati onas well as in insulin infusion pumps or artificia lpancreas closed-loop systems.

Properties of the copolymer compositions The polyacrylamide-based copolymers of the present disclosure have uniqu e properties and, when used in an aqueous formulati onwith a biologic molecule can, impart beneficial properties to the formulation.

In some embodiments, the copolymers described in the present disclosure reduce or prevent aggregation of a biologic molecule or lipid-based vehicle ,such as when formulat edin an aqueou sformulati oncontaining a biologic molecule or lipid-base d vehicle. In some embodiments, the biologic molecule is a protein. Thus, in some embodiments, the copolymers described in the present disclosure reduc eor prevent protein aggregation, such as when formulated in an aqueou sformulati oncontaining the protein.

The copolymers can be used with any protein that is susceptible to aggregation in an aqueous medium .Non-limiting examples include antibodie sand fragment sthereof, cytokines ,chemokines, hormones, vaccine antigens, cancer antigens, adjuvants and, combinations thereof. In some embodiments, the protein is a monoclonal antibody. In some embodiments, the polyacrylamide-bas edcopolymers reduce or prevent aggregation of a protein susceptib leto aggregation in an aqueou smedium. In some embodiments, the protein is insulin, or an analog thereof. In some embodiments, the protein is a monoclonal antibody. In some embodiments, the protein is a hormone. In some embodiments, the protein is a vaccine.

The copolymers of the present disclosure can also increase the stabilit ofy a formulation. According to embodiments of the disclosure the, copolymers described herein can be added to improve the stabilit ofy a formulati oncontaining, for example, a biologic molecule or a lipid-based vehicle for, example, peptides, proteins ,and conjugates thereof, nuclei cacids and oligonucleotide liposs, omes polymer, osomes mice, lles and, lipid nanoparticles. In some embodiments, additio nof a copolymer of the present disclosure increases the stabilit ofy a protein formulation. In some embodiments, additio nof a copolymer of the present disclosure increases the stabilit ofy a liposome formulatio n.In some embodiments, additio nof a copolymer of the present disclosure increases the stabilit ofy a micelle formulation. In some embodiments, additio nof a copolymer of the present disclosure increases the stabilit ofy a lipid nanoparticle formulation. In some embodiments, the additio nof a copolymer of the present disclosure increases the stabilit y of a formulati oncomprising one or more nuclei cacids. In some embodiments the, addition of a copolymer of the present disclosure increases the stabilit ofy a formulati oncomprising one or more messenger RNA (mRNA). In some embodiments the, addition of a copolymer of the present disclosure increases the stabilit ofy a formulati oncomprising one or more smal linterfering RNA (siRNA). In some embodiments, the additio nof a copolymer of the present disclosure increases the stabilit ofy a formulation comprising one or more deoxyribose nuclei cacids (DNA).

In some embodiments the, copolymers of the present disclosure increase the thermal stabilit ofy a formulati oncontaining a biologic molecule whic, h, in some embodiments, is a protein. Protein formulations typically require costl yrefrigerated transport and storage to prevent los sof protein integrity. Maintaining protein integrit yis a challenge for the pharmaceutical industry, health care providers, and patients worldwide, particularl iny the developing and low income regions where cold chain required for maintaining protein potency and efficacy is imperfect, overburdened or nonexistent. This results in large amounts of therapeutic protein formulations being wasted and potential ly endangering the live sof patients Thus,. interruptions in the cold chain can be costl y.The copolymer sof the present disclosure can imbue long-term stabilit and/ory cold chain resilience to protein formulations For. example, whil ecommercial protein formulations have good shelf live swhen stored properly (i.e., refrigerated), interruptions in the cold chain can decrease protein bioactivity and formulati onintegrity.

Protein formulations containing the copolymers of the present disclosure are, in some embodiments, stable for extende dperiods of time at temperatures higher than what is typically required for cold chain storage. For example, protein formulations containing the copolymers of the present disclosure can be stored at room temperature (between about °C to about 22 °C) or at elevated temperature suchs, as about 25 °C, about 30 °C, about °C, about 40 °C, about 45 °C, or higher while maintaining protein integrity. In some embodiments, protein formulations containing the copolymers of the present disclosure can be stored at a higher temperature than typically required, such as at about -20 °C, or about 0 °C, or about 2 °C, or about 4 °C, or about 8 °C, or about 20 °C, instead of at a lower temperature, such as at about -80 °C, or about -60 °C, or about -40 °C. In some embodiments, the copolymer senable protein formulations to be stored at room temperature or higher temperatures rather than requiring cold chain storage. In some embodiments, cold chain storage is not required to maintain protein integrity.

In some embodiments use, of the copolymers of the present disclosure in protein formulations maintains formulati onintegrity, bioactivit y,pharmacokinetics , and pharmacodynamics over a longer period of time when exposed to conditions such as elevated temperatures and agitation as compared to the same formulation without the copolymer. In some embodiments, the formulations maintain protein integrit yfor about 1 day, about 1 week, about 1 month, about 3 months, about 6 months, about 9 months, about 12 months, about 18 months, about 24 months, or longer at higher temperatures as compared to the same formulati onthat does not contain the copolymer of the present disclosure.

In some embodiments, the copolymers of the present disclosure reduce the rate of aggregation of a biologic molecule in an aqueou sformulation. In some embodiments, the copolymer sof the present disclosure reduce the rate of aggregation of a protein in an aqueous protein formulation. In some embodiments, the protein is a protein that tends to aggregate in an aqueous medium. In some embodiments, the protein is an antibody, or fragment sthereof, a cytokine, a chemokine, a hormone, a vaccine antigen, a cancer antigen, an adjuvant, and combinations thereof. In some embodiments, this allows for formulati onof more concentrated protein solutions, thus reducing the volume of the protein solution that needs to be administered, for example, to a patient. In some embodiments, the composition comprises the protein in a concentration at least two times greater, at least three time sgreater, at least four time sgreater, or at least five time sgreater than the concentration of the same protein in the composition without the copolymer.

Thus, use of the copolymers of the present disclosure can imbue long-term stabilit y and/or cold chain resilience to protein formulations In. some embodiments, a protein in a composition that comprises a copolymer of the present disclosure exhibits increased stabilit wheny stored at room temperature as compared to a protein composition that does not contain the copolymer. In some embodiments, the increased stabilit isy at least 10- fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, or greater as compared to a protein composition that does not contain the copolymer. In some embodiments, the protein is insulin. In some embodiments ,the protein is a monoclonal antibody. In some embodiments, the protein is a hormone. In some embodiments the, protein is a vaccine.

Thus, in one particular embodiment, insulin in a composition that comprises a copolymer of the present disclosure exhibits increased stabilit wheny stored at room temperature as compared to an insulin composition that does not contain the copolymer.

In some embodiments the, increased stabilit isy at least 10-fold, at least 15-fold, at leas t -fold, at least 25-fold, at least 30-fold, at leas t35-fold, at least 40-fold, at least 45-fold, at least 50-fold, or greater as compared to an insulin composition that does not contain the copolymer.

In some embodiments, the copolymers of the present disclosure preserve protein activit throughy 6 months of stresse agind g without modifying one or more of formulati on pharmacokinetics protein, secondary structure, formulati onclarity, and in vivo bioactivity.

In some embodiments the, copolymer reduce sprotein aggregation in the formulati onwhen stored at ambient temperature (about 23 °C to about 27 °C) as compared to a protein formulati onthat does not contain the copolymer. In some embodiments, the copolymer reduce sprotein aggregation in the formulati onwhen stored at 37 °C as compared to a protein formulati onthat does not contain the copolymer. In some embodiments, the copolymer reduce sprotein aggregation in the formulati onwhen stored at 50 °C as compared to a protein formulati onthat does not contain the copolymer. In some embodiments, the protein is insulin. In some embodiments the, protein is a monoclonal antibody. In some embodiments, the protein is a hormone. In some embodiments, the protein is a vaccine.

In one particular embodiment, the copolymers of the present disclosure preserve insulin activit ythrough 6 months of stressed aging without modifying one or more of formulati onpharmacokinetics ,protein secondary structure, formulati onclarity, and in vivo bioactivit y.In some embodiments, the copolymer reduce sinsulin aggregation in the formulati onwhen stored at ambient temperature (about 23 °C to about 27 °C) as compared to a human insulin formulation that does not contain the copolymer. In some embodiments , the copolymer reduces insulin aggregation in the formulati onwhen stored at 37 °C as compared to a human insulin formulati onthat does not contain the copolymer. In some embodiments, the copolymer reduce sinsulin aggregation in the formulati onwhen stored at 50 °C as compared to a human insuli nformulation that does not contain the copolymer.

As described in Example 4, when subjected to harsh stresse daging test sin standar d packaging, HUMULIN® R formulat edwit hthe M0Ni23% copolymer did not aggregate for over 56 days when subject to constant agitation at 37 °C, and four days of constant agitation at 50 °C, whereas HUMULIN® R alone aggregated within two days at 37 °C and within one day at 50 °C. The stress aging conditions used in the studi esdescribed herein are designed to mimic typical storage and transportation conditions. Even in hot climate s wit hlimited cold chain infrastructure, it is unlikely that shipping containers woul remad in at 50 °C wit hcontinuous agitation with no reprieve for more than a 24-hour period.

In some embodiments the, copolymer increases the time to aggregation of a protein formulati onby at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at leas t -fold, at least 35-fold, at least 40-fold, at least 45-fold, or at least 50-fold when stored at 37 °C as compared to a protein formulation that does not contain the copolymer. Time to aggregation can be assessed by any known method, including ,for example, by a transmittanc assay.e In some embodiments, the copolymer increases the time to aggregation of a protein formulati onby at least 10-fold when stored at 37 °C as compared to a protein formulati onthat does not contain the copolymer. In some embodiments, the copolymer increases the time to aggregation of a protein formulation by at least 20-fold when stored at 37 °C as compared to a protein formulati onthat does not contain the copolymer. In some embodiments, the copolymer increases the time to aggregation of a protein formulati onby at leas t30-fold when stored at 37 °C as compared to a protein formulati onthat does not contain the copolymer. In some embodiments, the copolymer increases the time to aggregation of a protein formulation by at least 40-fold when stored at 37 °C as compared to a protein formulation that does not contain the copolymer. In some embodiments, the copolymer increases the time to aggregation of a protein formulati onby at least 50-fold when stored at 37 °C as compared to a protein formulati onthat does not contain the copolymer. In some embodiments, the copolymer increases the time to aggregation of a protein formulation by at least 10-fold, at leas t15-fold, at leas t20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at leas 40-fold,t at leas 45-fold,t or at leas t 50-fold when stored at 50 °C as compared to a protein formulati onthat does not contain the copolymer. In some embodiments, the copolymer increases the time to aggregation of a protein formulati onby at least 10-fold when stored at 50 °C as compared to a protein formulati onthat does not contain the copolymer. In some embodiments, the copolymer increases the time to aggregation of a protein formulation by at least 20-fold when stored at 50 °C as compared to a protein formulation that does not contain the copolymer. In some embodiments, the copolymer increases the time to aggregation of a protein formulati onby at least 30-fold when stored at 50 °C as compared to a protein formulati onthat does not contain the copolymer. In some embodiments, the copolymer increases the time to aggregation of a protein formulati onby at least 40-fold when stored at 50 °C as compared to a protein formulati onthat does not contain the copolymer. In some embodiments the, copolymer increases the time to aggregation of a protein formulation by at least 50-fold when stored at 50 °C as compared to a protein formulati onthat does not contain the copolymer. In some embodiments, the protein is insulin. In some embodiments, the protein is a monoclonal antibody. In some embodiments, the protein is a hormone. In some embodiments, the protein is a vaccine.

In one particular embodiment, the copolymer increases the time to aggregation of the human insulin formulati onby at least 10-fold, at least 15-fold, at least 20-fold, at leas t -fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, or at leas t50- fold when stored at 37 °C as assessed by a transmittanc asse ay as compared to a human insulin formulation that does not contain the copolymer. In some embodiments the, copolymer increases the time to aggregation of the human insulin formulati onby at leas t -fold when stored at 37 °C as assessed by a transmittanc asse ay as compared to a human insulin formulati onthat does not contain the copolymer. In some embodiments the, copolymer increases the time to aggregation of the human insulin formulati onby at leas t -fold when stored at 37 °C as assessed by a transmittanc asse ay as compared to a human insulin formulati onthat does not contain the copolymer. In some embodiments the, copolymer increases the time to aggregation of the human insulin formulati onby at leas t -fold when stored at 37 °C as assessed by a transmittanc asse ay as compared to a human insulin formulati onthat does not contain the copolymer. In some embodiments, the copolymer increases the time to aggregation of the human insulin formulati onby at leas t 40-fold when stored at 37 °C as assessed by a transmittanc asse ay as compared to a human insulin formulati onthat does not contain the copolymer. In some embodiments, the copolymer increases the time to aggregation of the human insulin formulati onby at leas t 50-fold when stored at 37 °C as assessed by a transmittanc asse ay as compared to a human insulin formulati onthat does not contain the copolymer. In some embodiments, the copolymer increases the time to aggregation of the human insulin formulati onby at leas t -fold, at least 15-fold, at least 20-fold, at leas t25-fold, at least 30-fold, at least 35-fold, at leas t40-fold, at least 45-fold, or at leas t50-fold when stored at 50 °C as assessed by a transmittanc asse ay as compared to a human insulin formulati onthat does not contain the copolymer. In some embodiments the, copolymer increases the time to aggregation of the human insulin formulati onby at least 10-fold when stored at 50 °C as assessed by a transmittanc asse ay as compared to a human insulin formulati onthat does not contain the copolymer. In some embodiments the, copolymer increases the time to aggregation of the human insulin formulati onby at least 20-fold when stored at 50 °C as assessed by a transmittanc asse ay as compared to a human insulin formulati onthat does not contain the copolymer. In some embodiments the, copolymer increases the time to aggregation of the human insulin formulati onby at least 30-fold when stored at 50 °C as assessed by a transmittanc asse ay as compared to a human insulin formulati onthat does not contain the copolymer. In some embodiments the, copolymer increases the time to aggregation of the human insulin formulati onby at least 40-fold when stored at 50 °C as assessed by a transmittanc asse ay as compared to a human insulin formulati onthat does not contain the copolymer. In some embodiments the, copolymer increases the time to aggregation of the human insulin formulati onby at least 50-fold when stored at 50 °C as assessed by a transmittanc asse ay as compared to a human insulin formulati onthat does not contain the copolymer.

The increased stabilit obsery ved for HUMULIN® R formulated wit hthe M0Ni23% copolymer compared to HUMULIN® R alone suggests that the M0Ni23% copolymer has utility in stabilizing insulin under various agitation conditions where interfacial turnover may be higher (i.e., horizontal agitation). The conditions evaluated in Exampl e4 of the present disclosure represent extreme exposure conditions during shipping in uninsulate d containers or trucks in the hottes climt ates in the world where transport can take weeks before reaching patients.

In additio nto refrigerated transport in the early stages of the cold chain, maintaining proper transport and storage conditions during local distribut ionand once in patients’ hands presents a challenge in many parts of the world. As described herein, the addition of the polyacrylamide-based copolymers can preserve protein formulati on integrit yduring even severe cold chain interruptions In. some embodiments, this enables a reduction in cold chain requirements for protein transportation and storage that are difficul tto maintain in under-resourced environment s.As disclose dherein, the polyacrylamide-bas edcopolymer sas formulati onadditive scan improve cold chain resilience, thereby expanding global access to critical drugs and vaccines. In some embodiments, addition of the copolymer maintains the in vitro bioactivity of a protein formulati onfor at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, or at least 6 months. In some embodiments addi, tio nof the copolymer maintains the in vitro bioactivity of a protein formulati onfor at least 1 month. In some embodiments, addition of the copolymer maintains the in vitro bioactivity of a protein formulati onfor at least 2 months. In some embodiments addi, tio nof the copolymer maintains the in vitro bioactivity of a protein formulati onfor at least 3 months. In some embodiments, the protein is insulin. In some embodiments the, protein is a monoclonal antibody. In some embodiments, the protein is a hormone. In some embodiments, the protein is a vaccine.

In some embodiments, the copolymer present in a formulati onwith a protein does not alter the pharmacokinetic (PK) or pharmacodynamic (PD) properties of the active ingredients within the formulation.

Methods of using the copolymer compositions Provided in the present disclosure are methods of using the compositions and co- formulations described in the present disclosure that contain a polyacrylamide-based copolymer containing a water-soluble carrier monomer and a functional dopant monomer.

Provided in the present disclosure are methods of managing the blood glucose level in a subject in need thereof. In some embodiments, provided is a method of treating elevated blood glucose level ins a subject in need thereof. In some embodiments, the elevated blood glucose leve lis associated wit hinsulin deficiency. In some embodiments , the elevated blood glucose leve isl associated wit hintake of food (e.g., during a mealtime).

For example, provided is a method to offset the rise in glucos levee ls that can accompany ingesting macronutrients that raise glucos e,such as carbohydrates (i.e., mealtim insulie n).

In some embodiments, managing the blood glucose leve lcomprises reducing the glucose level in a subject in need thereof. In some embodiments, managing the blood glucose level comprises regulating the blood glucose leve lin a subject diagnosed wit hType 1 or Type 2 diabetes. In some embodiments mana, ging the blood glucose leve lcomprises reducing glucose level durings mealtime The. term "insulin deficiency," as used herein, refers to reduced insulin levels and/or reduced insulin sensitivit relaty ive to metabolic demand. A subject wit hinsuli ndeficiency includes, but is not limited to, a subject wit hdiabetes, including ,but not limited to, Type 1 diabetes, Type 1.5 diabetes, Type 2 diabetes, gestational diabetes mellitus, and diabetes post-pancreatectomy, a subject wit h hyperglycemia, and a subject wit h transient hyperglycemia , such as transient hyperglycemi afrom stress in an otherwise non-diabetic subject, for example, during hospitalizatio Inn. humans ,blood sugar level lesss than 100 mg/dL after fasting for at leas t eight hours and less than 140 mg/dL two hours after eating are deemed normal. A human subject wit hthe normal blood sugar levels described above are considered to be non- diabetic. The methods of the present disclosure include administering to a subject in need thereof a therapeuticall effecty ive amount of a composition of the present disclosure comprising a polyacrylamide-bas edcopolymer and insulin, or an analog thereof. In some embodiments, the composition is administered via a pump. In some embodiments, the pump is an infusion pump. In some embodiments the, composition is administere viad an artificial pancreas closed-loop system. In some embodiments, the composition is administered via an automat edinsulin delivery system. In some embodiments, the elevated glucose leve lis associated wit hinsulin deficiency in the subject. In some embodiment s, the subject has been diagnosed with Type 1 diabetes. In some embodiments, the subject has been diagnosed with Type 2 diabetes. In some embodiments, the subject is non- diabetic. In some embodiments, the subject has experienced trauma, surgery ,or both.

Also provided is a method for increasing stabilit ofy a formulati oncontaining a biologic molecule or lipid-based vehicle .According to embodiments of the disclosure the, copolymer sdescribed herein can be added to improve the stabili tyof formulations comprising peptides, proteins ,and conjugates thereof, nuclei cacids and oligonucleotide s, liposomes polymer, osomes mice, lles and, lipid nanoparticles. In some embodiments , provided is a method for increasing stabilit yof a protein formulation. In some embodiments, provided is a method of increasing thermal stabilit ofy a formulati on containing a biologic molecule or lipid-based vehicle. In some embodiments, provided is a method for increasing thermal stabilit ofy a protein formulation. Also provided is a method of reducing the rate of aggregation of a biologic molecule or lipid-based vehicl e in an aqueou sformulation. In some embodiments, the molecule is a molecule that tends to aggregate in an aqueous medium. In some embodiments, the protein is an antibody, or fragment sthereof, a cytokine, a chemokine, a hormone, a vaccine antigen, a cancer antigen, an adjuvant, and combinations thereof.

In some embodiments the, methods include adding about 0.001 wt% to about 5 wt% of the polyacrylamide-base copolymerd of the present disclosure to the formulati on containing a biologic molecule or lipid-based vehicle ,such as adding about 0.001 wt% to about 4 wt%, about 0.001 wt% to about 3 wt%, about 0.001 wt% to about 2 wt%, about 0.001 wt% to about 1 wt% ,about 0.001 wt% to about 0.5 wt%, about 0.001 wt% to about 0.4 wt%, about 0.001 wt% to about 0.3 wt%, about 0.001 wt% to about 0.2 wt%, about 0.001 wt% to about 0.1 wt%, about 0.001 wt% to about 0.05 wt%, about 0.001 wt% to about 0.02 wt%, about 0.001 wt% to about 0.01 wt%, about 0.001 wt% to about 0.005 wt%, about 0.005 wt% to about 5 wt%, about 0.005 wt% to about 4 wt%, about 0.005 wt% to about 3 wt%, about 0.005 wt% to about 2 wt%, about 0.005 wt% to about 1 wt%, about 0.005 wt% to about 0.5 wt%, about 0.005 wt% to about 0.4 wt%, about 0.005 wt% to about 0.3 wt%, about 0.005 wt% to about 0.2 wt%, about 0.005 wt% to about 0.1 wt%, about 0.005 wt% to about 0.05 wt%, about 0.005 wt% to about 0.02 wt%, about 0.005 wt% to about 0.01 wt%, about 0.01 wt% to about 5 wt%, about 0.01 wt% to about 4 wt% ,about 0.01 wt% to about 3 wt% ,about 0.01 wt% to about 2 wt% ,about 0.01 wt% to about 1 wt% about, 0.01 wt% to about 0.5 wt% about, 0.01 wt% to about 0.4 wt%, about 0.01 wt% to about 0.3 wt%, about 0.01 wt% to about 0.2 wt% ,about 0.01 wt% to about 0.1 wt%, about 0.01 wt% to about 0.05 wt% ,about 0.01 wt% to about 0.02 wt%, about 0.02 wt% to about 5 wt%, about 0.02 wt% to about 4 wt% ,about 0.02 wt% to about 3 wt%, about 0.02 wt% to about 2 wt%, about 0.02 wt% to about 1 wt%, about 0.02 wt% to about 0.5 wt%, about 0.02 wt% to about 0.4 wt% ,about 0.02 wt% to about 0.3 wt%, about 0.02 wt% to about 0.2 wt%, about 0.02 wt% to about 0.1 wt%, about 0.02 wt% to about 0.05 wt%, about 0.05 wt% to about 5 wt% ,about 0.05 wt% to about 4 wt% ,about 0.05 wt% to about 3 wt%, about 0.05 wt% to about 2 wt%, about 0.05 wt% to about 1 wt%, about 0.05 wt% to about 0.5 wt%, about 0.05 wt% to about 0.4 wt% ,about 0.05 wt% to about 0.3 wt%, about 0.05 wt% to about 0.2 wt%, about 0.05 wt% to about 0.1 wt%, about 0.1 wt% to about 5 wt%, about 0.1 wt% to about 4 wt%, about 0.1 wt% to about 3 wt% ,about 0.1 wt% to about 2 wt% ,about 0.1 wt% to about 1 wt% ,about 0.1 wt% to about 0.5 wt%, about 0.1 wt% to about 0.4 wt%, about 0.1 wt% to about 0.3 wt% ,about 0.1 wt% to about 0.2 wt%, about 0.2 wt% to about 5 wt%, about 0.2 wt% to about 4 wt%, about 0.2 wt% to about 3 wt%, about 0.2 wt% to about 2 wt%, about 0.2 wt% to about 1 wt% ,about 0.2 wt% to about 0.5 wt%, about 0.2 wt% to about 0.4 wt%, about 0.2 wt% to about 0.3 wt%, about 0.3 wt% to about 5 wt%, about 0.3 wt% to about 4 wt%, about 0.3 wt% to about 3 wt% ,about 0.3 wt% to about 2 wt%, about 0.3 wt% to about 1 wt% ,about 0.3 wt% to about 0.5 wt% ,about 0.3 wt% to about 0.4 wt% ,about 0.4 wt% to about 5 wt%, about 0.4 wt% to about 4 wt%, about 0.4 wt% to about 3 wt%, about 0.4 wt% to about 2 wt%, about 0.4 wt% to about 1 wt%, about 0.4 wt% to about 0.5 wt%, about 0.5 wt% to about 5 wt%, about 0.5 wt% to about 4 wt%, about 0.5 wt% to about 3 wt%, about 0.5 wt% to about 2 wt% ,about 0.5 wt% to about 1 wt%, about 1 wt% to about 5 wt%, about 1 wt% to about 4 wt%, about 1 wt% to about 3 wt% ,about 1 wt% to about 2 wt% about, 2 wt% to about wt%, about 2 wt% to about 4 wt% ,about 2 wt% to about 3 wt% about, 3 wt% to about wt%, about 3 wt% to about 4 wt%, about 4 wt% to about 5 wt%, or about 0.005 wt%, about 0.01 wt%, or about 0.1 wt% of the polyacrylamide-based copolymer of the present disclosure to the formulation. In some embodiments, the method includes adding about 0.0005 wt% to about 5 wt% of the polyacrylamide-base copolymerd of the present disclosure to the formulation. In some embodiments, the method includes adding about 0.001 wt% to about 1 wt% of the polyacrylamide-based copolymer of the present disclosure to the formulatio n.In some embodiments, the formulati oncomprises a biologic molecule. In some embodiments ,the formulati oncomprises a protein. In some embodiments, the formulati oncomprises a lipid-based vehicle. In some embodiments the, lipid-based vehicle comprises a liposome, lipid nanoparticle, polymerosome, or micelle.

Other uses of the polyacrylamide-based copolymers The polyacrylamide-based copolymers of the present disclosure can be used in any composition or formulati onwhere a surfactant is used. For example, the copolymers of the present disclosure can be used in applications including but, not limited to, cosmetic product s,hair product s,lotions, food products nutrit, ional product s,pigments and, ink. In some embodiments, copolymers are used to improve food texture or improve mouth-feel .

In some embodiments, the copolymers are used in biopharmaceutical compositions for animal or veterinary use. In some embodiments, the presence of the copolymer improves the stabilit ofy the formulation.

EXAMPLES Example 1 - Synthesi sand characterization of AD/DC copolymer library Study Design: The pharmacokinetic sof an insuli nlispro formulation, ultra-fast absorbing insulin lispro (UFAL), were compared to a commercial insulin lispro formulati on(HUMALOG®). Blood glucose and plasm alispro concentrations were measured afte rsubcutaneous administrati onof either (i) HUMALOG® or (ii) UFAL by using a handheld blood glucose monitor or ELISA on collecte dblood sample s.

Randomization: 5 pigs were used for this study and each pig received each formulati on once. The order in which the formulations were given in was randomized. Blinding: For analysis of pharmacokinetic parameters (t50% up, time to peak, t50% down) pharmacokineti c curves were coded, and were analyzed by a blinded researcher.

Replication. 5 pigs were used in this study and each pig acted as its own control receiving each formulati on(HUMALOG® and UFAL) once.

Materials: Solvents N,N-dimethy!formamide (DMF; HPLC Grade, Alfa Aesar, >99.7%), ethanol (EtOH; Certified ACS, Acros, >99.5%), acetone (Sigma, HPLC Grade, >99.9%), hexanes (Fisher, Certified ACS, >99.9%), ether (Sigma, Certified ACS, Anhydrous, >99%) and CDCl3 (Acros, >99.8%) were used as received .Monomers N,N- dimethylacrylamide (DMA; Sigma, 99%), N-(3-methoxypropyl)acrylami de(MPAM; Sigma, 95%), 4-acryloylmorpholine (MORPH; Sigma, >97%), acrylamide (AM; Sigma, >99%), and N-hydroxyethyl acrylamide (HEAM; Sigma, >97%) were filtered wit hbasic alumina prior to use. Monomers N-phenylacrylamide (PHE; Sigma, 99%), N-tert- butyl acrylamide (TEA; Sigma, 97%), N-isopropylacrylamide (NIP AM; Sigma, >99%), and N-[tris(hydroxymethyl)methyl]acrylami (TRIde; Sigma, 93%) were used as received. (3-acrylamidopropyl)trimethylammonium (TMA; Sigma, 75%) was washed wit hethyl acetate. 2-acrylamido-2-methylpropane sulfonic acid (AMP; Sigma, 99%) was converted to the sodium salt throug hequimolar mixing wit hsodium acetat ein methanol and precipitated into acetone . RAFT chain transfe r agents 2-cyano-2-propyl dodecyl trithiocarb onate (2-CPDT; Strem Chemicals, >97%) and 4-((((2- carboxyethyl)thio)carbonothioyl)thio)-4-cyanopenta noicacid (BM1433; Boron Molecular, >95%) were used as received .Initiator 2,2’-azobis(2-methyl-propionitril e) (AIBN; Sigma, >98%) was recrystallized from methanol (MeOH; Fisher, HPLC Grade, >99.9%) and dried under vacuum before use. Initiator 4,4-azobis(4-cyanovaleric acid)(ACVA; Sigma, >98%) was used as received. Z group removing agents lauroyl peroxide (LPO; Sigma, 97%) and hydrogen peroxide (H2O2; Sigma, 30%) were used as received.

Synthesi s of first copolymer library via automated parallel synthesis: Copolymerizations of carriers and dopants were carried out using RAFT polymerization ([Total Monomer]/[CTA] = 50, [CTA]/[AIBN] = 0.2). MP AM, MORPH, and DMA carrier monomers copolymerized wit hAMP, TMA, NIP, TBA, or PHE dopant monomers were polymerized in DMF using 2-CPDT as the CTA and AIBN as the initiator. MP AM, MORPH, and DMA carrier monomers copolymerized wit hTRI dopant monomer were polymerized in a DMF/water mixture using BM1433 as the CTA and ACVA as the initiator. Total vinyl monomer molarity was held at 2.72M (MPAM copolymerizations ), 2.86M (MORPH copolymerizations), and 3.84M (DMA copolymerizations) such that the homopolymerization of the carrier monomer in DMF woul dbe carried at a constant 40 wt.%. HEAM carrier monomer copolymerized with AMP, TMA, NIP, TBA, or PHE dopant monomers were polymerized in DMF/EtOH mixture using 2-CPDT as the CTA and AIBN as the initiator. HEAM carrier monomer copolymerized wit hAMP, TMA, NIP, TBA, or PHE dopant monomers were polymerized in DMF/EtOH/water mixture using BM1433 as the CTA and ACVA as the initiator. Total vinyl monomer molarit wasy held at 2.58M (HEAM copolymerizations) such that the homopolymerization of HEAM in DMF woul dbe carried at a constant 30 wt.%. AM carrier monomer copolymerized with AMP, TMA, NIP, TBA, or PHE dopant monomers were polymerized in DMF/wate r mixture using BM1433 as the CTA and ACVA as the initiator. AM carrier monomer copolymerized with TRI dopant monomer was polymerized in water using BM1433 as the CTA and ACVA as the initiator. Total vinyl monomer molarit ywas held at 4.05M (AM copolymerizations) such that the homopolymerization of AM in DMF woul bed carried at a constant 30 wt.%.

Reaction mixtures were prepared by combining stock solutions: (i) carriers, (ii) dopants, and (iii) CTA and initiator. The stock solutions of carrier monomers were HEAM (555 mg/mL in EtOH), AM (462 mg/mL in water), MP AM (818 mg/mL in DMF), DMA (no solvent dilution), and MORPH (no solvent dilution). The stock solutions of dopant monomers were TRI (181mg/mL in water), PHE (120 mg/mL in DMF), NIP (245 mg/mL in DMF), TBA (122 mg/mL in DMF), AMP (120 mg/mL in DMF), and TMA (124 mg/mL in DMF). Stock solutions of CTA and initiator were prepared such that [CTA]/[initiator ] = 5 for AM (BM1433 at 310 mg/mL in water), HEAM and MP AM (BM1433 at 198 mg/mL in water, and 2-CPDT at 221 mg/mL in DMF), MORPH (BM1433 at 220 mg/mL in water and 2-CPDT at 247 mg/mL in DMF), and DMA (BM1433 at 220 mg/mL in water and 2-CPDT at 332 mg/mL in DMF). Reaction mixtures of HEAM, DMA, MP AM, and MORPH were diluted wit hDMF while reaction mixtures of AM were dilut edwit hwater to reach the desired vinyl monomer concentration.

Parallel syntheses of polyacrylamide-bas edcopolymer excipient s(also referred to as AC/DC excipients) were conducted on a Chemspeed Swing XL automated synthesizer robot equipped wit ha 4-Needl eHead tool and an iSynth reactor. The Reactions were performed in 8 mL disposable ISynth reactor vials. All aspirations and dispensing reagent solutions were performed using a the 4-Needl eHead tool equipped wit ha 2 x 10 mL and 2x1 mL syringes fitted with septa piercing needles, wit hboth the 1 mL and 10 mL syringes used in this particular experiment. All solvent lines were primed wit h60 mL (6 strokes of syringe volum e)of degassed DMF. Typical aspiration and dispense rates of the reagents were 10 mL/min for both the 1 mL syringes .An airgap of 50 pL and an extra volume of 50 pL was used for the 1 mL syringes, and an airgap of 50 pL and an extra volume of 100 pL was used for the 10 mL syringes during aspirations using the 4-Needle Head tool The. needles and lines were rinsed after each reagent dispens etask wit h3 mL inside and outside volume of the priming solvent for the 1 mL syringes and wit h20 mL inside and outside volume of the priming solvent for the 10 mL syringes. The DMF reservoir was degassed by continuous nitrogen sparging. All stock solutions were prepared in septa capped reagent vial sand degasse dby sparging wit hargon for 15 minutes before transfer into the Chemspeed. The atmosphere withi nthe Chemspeed was reduced to <1 % oxygen by purging wit hnitrogen while exhaus tports were closed. Reactor vial swere exposed to nitrogen flow until the start of the reaction. The calculated aliquots of stock solutions and solvent were transferred to the reactors via the automated liquid handling system. Upon dispensing, reactor vial swere manually sealed in the inert atmosphere, removed from the Chemspeed, manuall shakeny to combine reagents, and heated to 65 °C in an oven for 24 hours, after which, reaction vial swere cooled to room temperatur ande exposed to air.

A procedur eto remove the CTA Z groups from the AC/DC excipient scontaining MORPH, DMA, HEAM, and MP AM copolymers was adapted from the literature. The reaction vial was dilut edto 6 mL wit hDMF. LPO (2 eq.) and AIBN (20 eq.) were added to the reaction mixture, which was sealed wit ha cap utilizing a PTFE seal .The reaction mixture was sparged wit hnitrogen gas for 10 minutes while heating at 90 °C and subsequently heated for 12 hours at 90 °C. A procedure to remove the CTA Z groups from the AC/DC excipient scontaining AM copolymers was adapted from the literatur Thee. reaction vial was diluted to 5 mL wit hmiliQ water. H2O2 (20 eq.) was added to the reaction vial ,which was sealed and heated to 60 °C for 12 hours. The resulting copolymer swere isolated by precipitation as outlined below.

AC/DC excipient ssynthesized wit hAM and HEAM carriers were precipitated twice from ace-tone. AC/DC excipient ssynthesized wit hDMA and MORPH were precipitated twice from diethyl ether . AC/DC excipient ssynthesized with MP AM were precipitated twice from diethyl ethe rand hexane (3:1 ratio) mixtures. The number (Mn) and weight (Mv) average molecula weigr hts and dispersity for the AC/DC excipients containing MORPH, MP AM, DMA, and HEAM were determined using SEC in DMF wit h poly(ethyleneglycol standar) ds. Mn, Mw, and dispersit yfor the AC/DC excipients containing AM were determined using aqueous SEC-MALLS.

Synthesi sof second polymeri clibrary: A typical procedure to synthesiz ea MORPH-NIP AC/DC excipient is as follows and is nearly identical for all other carrier/dopant combinations, where only the carrier/dopant selection and concentration are changed. MORPH (645 mg, 4.57 mmol ,41.5 eq.), NIP AM (105 mg, 0.93 mmol ,8.5 eq.), 2CPDT (38 mg, 0.11 mmol ,1 eq.) and AIBN (3.6 mg, 0.02 mmol, 0.2 eq.) were combined and diluted wit hDMF to a total volume of 2.25 mL (33.3 w/v vinyl monomer concentration) in an 8 mL scintillat ionvial equipped wit ha PTFE septa. The reaction mixture was sparged wit hnitrogen gas for 10 minutes and then heated for 12 hours at 65 °C. To remove the Z-terminus of the resulting polymer, AIBN (360 mg, 2.2 mmol 20, eq.) and LPO (88 mg, 0.22 mmol ,2 eq.) were added to the reaction mixture, which was then sparged wit hnitrogen gas for 10 minutes and heated for 12 hours at 90 °C. Z-group removal was confirmed by the ratio of the refractive index to UV (X = 310 nm) intensity in SEC analysis. Resulting polymers were precipitated three time sfrom ether ,and dried under vacuum overnight. Resulting composition and molecula weigr hts were determine d via 1H NMR spectroscopy and SEC with poly(ethyleneglycol sta) ndards.

Copolyme rmolecul arweight characterization: Mn, Mw, and dispersity for copolymer swith HEAM, DMA, MP AM, and MORPH carrier monomers were determine d via SEC implementing poly(ethyleneglycol sta) ndards (American Polyme rStandards Corporation) after passing through two size exclusion chromatography columns (Re-solve Mixed Bed Low DVB, ID 7.8 mm, Mv range 200-600,000 g mol'l (Jordi Labs) in a mobile phase of N,N-dimethylformamid (DMFe ) wit h0.1M LiBr at 35 °C and a flow rate of 1.0 ml min1־ (Dionex Ultimate 3000 pump, degasser, and autosampler (Thermo Fisher Scientific).

Mn, Mw, and dispersit yfor copolymers wit hAM were determined via SEC- MALLS after passing through a size exclusion chromatography column (Superose 6 Increase 10/300 GL, 5,000-5,000,000 g mol1־ (GE Healthcare)) in a mobile phase of phosphate-buffered saline containing 300 ppm sodium azide. Detection consiste dof a Optilab T-rEX (Wyatt Technology Corporation) refractive index detector operating at 658 nm and a TREOS II light scattering detector (Wyatt Technology Corporation) operating at 659 nm. The dn/dc value for AM copolymers were assumed to be 0.185 in this media.

Methodfor Determining Experimental VM wt. % Vallies The handling of viscous monomers (HEAM, MP AM, MORPH) by the Chemspeed resulted in monomer loadings that differed from the target monomer loadings for select copolymerizations during the initial AC/DC copolymer library synthesis Experi. mental weight percentages were approximated from the peak molecula weigr hts (Mp) of the SEC traces. Because only viscous monomers were affected, changes to Mp arose from inadequate addition of carrier monomer (aside from small changes in pervaded volume in the differing weight percentages of the dopant monomer compared to the carrier). Thus , to calculat thee experimental weight percentages, A/P,max was determined for a given carrier/dopant pair.

Results High-throughput synthesis of polyacrylamide library: A library of AC/DC excipient swas synthesized combinatoriall throughy statistical copolymerizations of water- solubl ecarrier monomers and functional dopant monomers (FIG. 2). The carrier monomers were the predominant species and responsible for both maintaining solubil ity and providing an inert barrier to prevent insulin-insulin interactions. The functional dopants copolymerize dat lower weight percentages were incorporated statistica lly throughout the resulting copolymer. These dopants are selected by design to promote either polymer-interface interactions or polymer-insulin interactions. The library target sa degree of polymerizatio n(DP) of 50 for the copolymers result, ing in molecul arweights similar to insulin and well below the glomeria lfiltratio thresholdn for synthet icpolymers.

The experimental degree of polymerization (DP) of the carrier monomer was approximated using equation (SI). This valu wase used to approximat ethe experimental weight percentages (wt%). Resul tscalculated using this method were corroborated by 1H NMR spectroscopy for the copolymerizations of MORPH (carrier) and PHE (dopant )as shown in FIG. 3. The results from this comparison are shown in Table 1. The experimental weight percentage values of the carrier monomer and the functional dopant monomer for each copolymer were determined using 1H NMR and SEC and are summarized in Table 2. ? ?carrier,experimental — 77 * ??carrier,target (S1) Mp,max Table 1. Validation of SEC wt.% measurement by 1HNMR Spectroscopy Carrier wt.% Dopant wt.% wt.%a wt.%b Mp (Target) (Target) (Experimental, NMR) (Experimental, Mp) MORPH 96.7 PHE 3.3 4.5 3.6 2900 MORPH 93.3 PHE 6.7 11.7 10.7 1850 MORPH 90 PHE 10 11.3 10 3100 a Experimental wt.% calculated from post precipitated 1H NMR (5 = 3.3-3.7, 8H). b Experimental wt.% calculated from equation 1 using Mp (peak molecular weight) values determined with SEC.

Table 2. SEC and MALS characterization and analysis of polymers synthesized in initial AC/DC library Carrier wt.% wt.% Dopant wt.% wt.% M״a Mwa Da (Target) (Experimental) (Target) (Experimental) (Da) (Da) DMA 100 - 0 - 2700 3000 1.1 DMA 93.34 - NIP 6.66 - 2900 3500 1.2 DMA 86.67 - NIP 13.33 - 3000 3500 1.15 DMA 80 - NIP 20 - 3000 3400 1.14 DMA 96.67 - PHE 3.33 - 2800 3200 1.14 DMA 93.34 - PHE 6.66 - 3000 3500 1.17 DMA 90 - PHE 10 - 3400 3900 1.15 DMA 96.67 - AMP 3.33 - 3400 4100 1.22 DMA 93.34 - AMP 6.66 - 3700 4400 1.2 DMA 90 - AMP 10 - 3500 4100 1.16 DMA 96.67 - TMA 3.33 - 3700 4300 1.15 DMA 93.34 - TMA 6.66 - 3800 4600 1.2 DMA 90 - TMA 10 - 3800 4500 1.19 DMA 96.67 - TEA 3.33 - 2900 3500 1.2 DMA 93.34 - TEA 6.66 - 3000 3600 1.2 DMA 90 - TEA 10 - 3100 3600 1.17 DMA 95 - TRI 5 - 2900 3500 1.2 DMA 90 - TRI 10 - 3500 4100 1.17 DMA 85 - TRI 15 - 3200 3900 1.23 MORPH 100 - 0 - 2300 2300 1.12 MORPH 93.34 90.2 NIP 6.66 9.8 1600 1800 1.12 MORPH 86.67 86.7 NIP 13.33 13.3 2300 2600 1.14 MORPH 80 78.7 NIP 20 21.3 2200 2500 1.13 MORPH 96.67 96.4 PHE 3.33 3.6 2500 2800 1.1 MORPH 93.34 89.3 PHE 6.66 10.7 1700 2000 1.16 MORPH 90 90 PHE 10 10 2700 3200 1.17 MORPH 96.67 95.6 AMP 3.33 4.4 2900 3300 1.14 MORPH 93.34 91.8 AMP 6.66 8.2 3000 3500 1.16 MORPH 90 90 AMP 10 10 3600 4200 1.17 MORPH 96.67 n/a TMA 3.33 n/a n/a n/a n/a MORPH 93.34 78.2 TMA 6.66 21.8 3700 4400 1.2 MORPH 90 90 TMA 10 10 1700 1900 1.1 MORPH 96.67 94.5 TEA 3.33 5.5 2400 2900 1.2 MORPH 93.34 92.8 TEA 6.66 7.2 2700 3300 1.2 MORPH 90 90 TEA 10 10 2900 3300 1.14 MORPH 95 92.7 TRI 5 7.3 2200 2500 1.14 Carrier wt.% wt.% Dopant wt.% wt.% M״a Mwa Da (Target) (Experimental) (Target) (Experimental) (Da) (Da) MORPH 90 90 TRI 10 10 3100 3900 1.25 MORPH 85 84.1 TRI 15 15.9 2900 3500 1.22 HE AM 100 - 0 - 4900 5500 1.13 HE AM 93.34 93 NIP 6.66 7 5300 6000 1.14 HE AM 86.67 86.7 NIP 13.33 13.3 5600 6300 1.13 HE AM 80 79.2 NIP 20 20.8 5400 6000 1.12 HE AM 96.67 96.7 PHE 3.33 3.3 5800 6600 1.14 HE AM 93.34 93 PHE 6.66 7 5500 6300 1.13 HE AM 90 89.4 PHE 10 10.6 5200 6100 1.16 HE AM 96.67 96.2 AMP 3.33 3.8 4900 5600 1.15 HE AM 93.34 93.3 AMP 6.66 6.7 5800 6600 1.12 HE AM 90 87.8 AMP 10 12.2 4800 5500 1.14 HE AM 96.67 96.2 TMA 3.33 3.8 5100 5900 1.15 HE AM 93.34 93.3 TMA 6.66 6.7 5500 6500 1.17 HE AM 90 86 TMA 10 14 3900 4500 1.15 HE AM 96.67 96.6 TEA 3.33 3.4 5300 6000 1.13 HE AM 93.34 93.3 TEA 6.66 6.7 5300 6100 1.14 HE AM 90 88.6 TEA 10 11.4 4700 5300 1.13 HE AM 95 95 TRI 5 5 5100 6000 1.17 HE AM 90 89.2 TRI 10 10.8 4600 5400 1.18 HE AM 85 84.3 TRI 15 15.7 4600 5400 1.18 MP AM 100 - 0 - 3600 4000 1.13 MP AM 93.34 93.3 NIP 6.66 6.7 4600 5100 1.12 MP AM 86.67 84.4 NIP 13.33 15.6 3800 4300 1.14 MP AM 80 72.5 NIP 20 27.5 3000 3300 1.12 MP AM 96.67 96.5 PHE 3.33 3.5 5000 5300 1.11 MP AM 93.34 93.3 PHE 6.66 6.7 5000 5700 1.14 MP AM 90 86.7 PHE 10 13.3 3900 4400 1.13 MP AM 96.67 96.3 AMP 3.33 3.7 4500 5100 1.14 MP AM 93.34 93.3 AMP 6.66 6.7 4900 5500 1.13 MP AM 90 87.9 AMP 10 12.1 4000 4500 1.13 MP AM 96.67 96.7 TMA 3.33 3.3 4600 5200 1.14 MP AM 93.34 91.9 TMA 6.66 8.1 3600 4100 1.13 MP AM 90 89.8 TMA 10 10.2 4400 5000 1.13 MP AM 96.67 96.6 TEA 3.33 3.4 4500 5100 1.13 MP AM 93.34 93.3 TEA 6.66 6.7 4800 5400 1.13 Carrier wt.% wt.% Dopant wt.% wt.% M״a Mwa Da (Target) (Experimental) (Target) (Experimental) (Da) (Da) MP AM 90 89.2 TEA 10 10.8 4200 4700 1.12 MP AM 95 93.9 TRI 5 6.1 4900 5700 1.17 MP AM 90 90 TRI 10 10 5800 6800 1.17 MP AM 85 80.3 TRI 15 19.7 4300 4800 1.13 AM 100 - 0 - 4800 5100 1.06 AM 93.34 - NIP 6.66 - 4400 4600 1.05 AM 86.67 - NIP 13.33 - 4500 4800 1.05 AM 80 - NIP 20 - 4800 5100 1.07 AM 96.67 - PHE 3.33 - 4300 4500 1.04 AM 93.34 - PHE 6.66 - 4600 4700 1.04 AM 90 - PHE 10 - 4500 4600 1.03 AM 96.67 - AMP 3.33 - 3700 4000 1.09 AM 93.34 - AMP 6.66 - 4000 4300 1.07 AM 90 - AMP 10 - 4100 4300 1.05 AM 96.67 - TMA 3.33 - 4300 4500 1.04 AM 93.34 - TMA 6.66 - did not elute AM 90 - TMA 10 - 4700 4900 1.06 AM 96.67 - TEA 3.33 - 4300 4400 1.04 AM 93.34 - TEA 6.66 - 4100 4300 1.03 AM 90 - TEA 10 - 4400 4600 1.05 AM 95 - TRI 5 - 4300 4500 1.05 AM 90 - TRI 10 - 4600 4800 1.04 AM 85 - TRI 15 - 4600 4900 1.06 a Mn (number average molecular weight), Mw (weight average molecular weight), and D (dispersity) determined via DMF size exclusion chromatography calibrated using polyethylene glycol standards for HEAM, MP AM, MORPH, and DMA. Mn and Mw determined using aqueous SEC-MALS for AM using a dn/dc value of 0.185. b Experimental wt.% values determined wit hSEC using equation S1.

The library was generated through parallel synthesis wit ha Chemspeed Swing XL Aut oSynthesizer, a liquid handling robot in an inert environment. RAFT polymerization was implemented because it affords precise copolymerization stoichiometry, low dispersit y,and controll edmolecula rweights for a wide scope of monomers.

Polyacrylamide derivatives were used for both the carrier and dopant monomers due to the scope and availabili tyof commerciall avaiy labl watere soluble monomers (carriers) and functional monomers (dopants) and polymeri cstability. While monomeric acrylamide derivatives often exhibit acute toxicities polyac, rylamide derivatives, when properly purified from their monomeric precursors demonstra, tea high degree of biocompatibility.

Moreover, the reactivit ratiosy between the variou sacrylamide monomers are close to 1, yielding copolymer swit hlittle to no dopant gradient composition. Carrier monomers included acrylamide (AM), hydroxyethylacrylami de(HEAM), dimethylacrylami de (DMA), acryloylmorpholi ne(MORPH), and methoxypropyl acrylamide (MP AM) as they are nonionic and water solubl (orderede in increasing hydrophobicity). Dopant monomers included tris(hydroxymethyl)methylacryla (TRI)mide, acrylamidomethylpropane sulfonic acid (AMP), acrylamidopropyltrimethylammonium chloride (TMA), n- isopropyl acrylamide (NIP) tertbutyl acrylamide (TEA), and phenyl acrylamide (PHE).

These functional dopants coul dbe further classified into hydrogen bonding (TRI), ionic (AMP, TMA), hydrophobic (NIP, TEA), and aromati c(PHE) monomers based on thei r chemical composition.

A library of 90 AC/DC excipient swere synthesized through the combinatorial copolymerization of carrier and dopant monomers at each of three different compositions for a given carrier-dopant pair. NIP was copolymerized at either 6.7, 13.3, or 20 wt.% .TRI was copolymerized at either 5, 10, or 15 wt.% .AMP, TMA, TEA, and PHE were copolymerized at either 3.3, 6.7, or 10 wt.%. These values were selected to maximize dopant loading while yielding functional copolymers wit hlower critical solution temperature (LCST) values above 37 °C to ensure they woul remaid n solubl ate all relevant temperatures. Polymers were characterized by NMR and SEC (Table 2, FIG. 3 and FIG. 4). While RAFT polymerizatio naffords many synthetic advantages, it yield spolymers wit ha reactive trithiocarbonate Chain Transfer Agent (CTA) attached at the Z-terminus.

Accordingly, the CTA moiety on the synthesized AC/DC excipient swas removed prior to utilizati onof the copolymers in subsequent assays to ensure their inertness.

Example 2 - Ultra-fast absorbing insulin lispro (UFAL) formulati onand in vitro and in vivo evaluations Method for Determining Mammalia nCel lViability: NIH/3T3 mouse fibroblasts from ATCC were cultured in DMEM containing 10 wt.% FES and 1 wt.% Penicillin- Streptomycin in a 37 °C, 5% CO2 incubator. 3T3s at passage 9 were seeded at 5000 cell s per well in a 96 wel lplate and cultured for 24 h in 100 pL of media. The media was subsequently replaced wit h100 pL of media containing MORPH-NIP23% at various concentrations and incubated for 24 h. The polymer-containing media was then aspirated from each well. Each well was then washed wit h100 pL of PBS and charged wit hboth 100 pL of new media and 10 pL of WST reagent. After 3 hours of incubation in the WST solution, the absorbance was read using a plat readere (X = 450nm). All experiment swere conducted in triplicate. Cel lviability was calculated using equation S2, where Awell, ^control , and v4wst are the absorbance measurements for the cell scultured wit hpolymer, the cell s cultured without polymer, and WST in media.

Viability = Awell־AwsT (82) Acontrol־AWST In vitro insulin cellular activity assay: C2C12 mouse muscle myoblasts (ATCC CRL-1772) were cultured to confirm insulin functional activit yvia the AKT phosphorylation pathway using AlphaLISA SureFire Ultra(Perkin-Elmer) kits for detection of phosphorylated AKT 1/2/3 (pS473) compared to total Aktl .Cell swere confirmed to be free of mycoplasm acontamination prior to use. Dulbecco’s Modified Eagle’s Medium (DMEM) wit h4.5 g/L D-glucose L-glut, amine, and 110 mg/L sodium pyruvate (Gibco) was supplemented wit h 10% fetal bovine serum (FBS) and 5% penicillin-streptomycin to formulat come plet eculture media. Cells were seeded at a density of 25,000 cells/we inll a volume of 200 uL/well in a 96-well tissue culture plat e and grown for 24 hours. Prior to insulin stimulati on,the cell weres washed twice wit h200 pL of unsupplemented DMEM and starved in 100 pL of unsupplemented DMEM overnight. The media was then removed and the cell swere stimulated with 100 pL of insulin (i) HUMALOG®, (ii) UFAL, (iii) Aged HUMALOG® (12 h shaking at 37 °C), (iv) Aged UFAL (12 h shaking at 37 °C) diluted in unsupplemented DMEM to the desired concentration, for 30 min while incubating at 37 °C. Cell weres washed twice wit h100 pL of cold IX Tris-buffered saline before adding 100 pL of lysis buffer to each wel land shaking for at least 10 minutes at room temperature to fully lyse cells. 30 pL of lysat wase transferred to a 96-wel lwhite half-area plate for each assay. Assays were complete d according to the manufacturer’s protocol. Plates were incubated at room temperatur ande read 18-20 hours after the addition of the final assay reagents using a Tecan Infinite Ml 000 PRO plat ereader. Resul tswere plotted as a ratio of [pAKT]/[AKT] for each sampl e(n=3 cellular replicates) and an EC50 regression [log(agonist) vs. response (three parameters)] was plotted using GraphPad Prism 8.

In vitro insulin stabilit y:Methods for aggregation assays for recombinant human insulin were adapted from Webber et al. (Proc. Natl. Acad. Set. U. S. A. 113, 14189-14194 (2016)). Briefly ,formulati onsamples (3.4 mg/mL) were plated at 150 pL per well (n = 3/group) in a clear 96-wel lplat eand sealed with optically clear and thermall sty abl seale (VWR). The plat ewas immediatel placy ed into a plat ereader and incubated wit h continuous shaking at 37°C. Absorbance readings were taken every 10 minutes at 540 nm for 100 h (BioTek Syner-gyHl microplat reade er). The aggregation of insulin leads to light scattering, which results in an increase in the measured absorbance. The time-to- aggregation (Za ) was defined as the time at which a greater than 10% increase in absorbance from the absorbance at time zero was observed. After 100 h, the plate was removed from the plat ereader and transferred to an incubator shaker plat ewhere it was subjected to continued stressed aging. Absorbance readings were taken periodically for up to 30 days.

For the initial high-throughpu stt abilit screy en, recombinant human insulin (Gibco) was formulated in phosphate buffered saline (0.9 wt.% NaCl )and AC/DC excipients were added at concentrations of 1 mg/mL or 10 mg/mL to the recombinant insuli nformulation for a final insulin concentration of 3.4 mg/mL. Each plate contained a recombinant insuli n contro lwit hno polymer added.

For the secondary stabilit screy en with UFAL formulations contro, lgroups included: (i) commercial HUMALOG® (Eli Lilly), (ii) zinc-free lispro comprising phosphate buffer, glycerol (2.6 wt.%), and phenoxyethanol (0.85 wt.%). Zinc (II) was removed from commercial insulin formulations through competitive binding by addition of ethylenediaminetetraaceti acidc (EDTA), which exhibits a dissociation binding constant approaching attomolar concentrations (Ad ~ 1018־ M). EDTA was added to formulations (1 eq. wit hrespect to zinc) to sequest erzinc from the formulation. Following zinc sequestration PD, MidiTrap G-10 gravity columns (GEHealthcare) were used to remove the zinc/EDTA complexe sand other formulati onexcipients. Lispro was concentrated using Amino Ultra 3K centrifugal units (Millipore), and then reformulated at 100 U/mL wit hphosphate buffer (lOmM), glycerol (2.6 wt.%), phenoxyethanol (0.85 wt.%), and AC/DC excipient (0.01 wt.%).

NMR POSY: 1H 2D DO SY spectra were recorded at an insulin lispro concentration of 3.4 mg/mL wit h40 wt.% D2O for UFAL formulati oncomprising phosphate buffer, glycerol (2.6 wt.%), phenoxyethanol (0.85 wt.%) and MORPH-NIP23% copolymer (0.1 wt.%). A Varian Inova 600 MHz NMR instrument was used to acquire the data. Magnetic field strengths ranging from 2 to 57 G cm1־. The DOSY time and gradient pulse were set at 132 ms (A) and 3 ms (5) respectively. All NMR data were processed using MestReNova 11.0.4 software.

Streptozotocin-induced model of diabetes in rats: Male Sprague Dawley rats (Charles River) were used for experiments. Animal studi eswere performed in accordance wit hthe guidelines for the care and use of laboratory animals; all protocols were approved by the Stanford Institutional Animal Care and Use Committee. The protocol used for STZ induction adapted from the protocol by Wu and Huan (Purr. Protoc. Pharmacol. (2008) :Unit 5.47). Briefly, mal eSprague Dawley rats 160-230 g (8-10 weeks) were weighed and fasted 6-8 hours prior to treatment wit hSTZ. STZ was diluted to 10 mg/mL in the sodium citrate buffer immediatel ybefore injection. STZ solution was injected intraperitoneall aty 65 mg/kg into each rat .Rats were provided wit hwater containing 10% sucrose for 24 hours after injection with STZ. Rat blood glucose level weres tested for hyperglycemi adail yafter the STZ treatment via a tail vein blood collection using a handheld Bayer Contour Next glucose monitor (Bayer). Diabetes was defined as having 3 consecutive blood glucose measurements >400 mg/dL in non-fasted rats.

In vivo pharmacodynamics in diabetic rats: Diabetic rats were fasted for 4-6 hours.

Rats were injected subcutaneously wit h (i) HUMALOG®, (ii) UFAL, (iii) Aged HUMALOG® (12h shaking at 37 °C), (iv) Aged UFAL (12 h shaking at 37 °C) at a dose of 1.5U/kg. To prepare aged samples 150, uL of each formulati onwas placed in a 96-well plat eunder constant agitation. 16 rats were used for this study and they were randomly assigned to two groups (i) HUMALOG® and (ii) UFAL. Within these groups each rat received one dose of the fresh and aged version of the formulati onon separate experimental days. The order that the formulations were given was randomized. Insulins were dilut ed10-fold in phosphate buffered saline before injection to allow for accurate dosing of smal lvolume s.Before injection, baseline blood glucose was measured After injection, blood was sampled every 30 minutes for 4 hours. Blood glucos wase measured using a handheld blood glucos monie tor (Bayer Contour Next).

Biocompatibility in diabetic rats: Diabetic rats were treated wit heither (i) HUMALOG® (n=5) or (ii) UFAL (n=5) for 7 consecutive days. Formulations were administered subcutaneously at a dose of 1.5 U/kg. Blood was collect edfor blood chemistry tests on day 0 and on Day 7. Chemistry analysis was performed on the Siemens Dimension Xpand analyzer . A medical technologist performed all testing, including dilutions and repeat test ass indicated, and reviewed all data.

In vivo pharmacokinetic sand pharmacokinetic sstudi esof ultra-fas abst orbing insulin lispro (UFAL) in Swine Model: Study Design: The pharmacokinetic sof an insulin lispro formulation, ultra-fast absorbing insulin lispro (UFAL), were compared to a commercial insulin lispro formulati on(HUMALOG®). Blood glucose and plasm alispro concentrations were measured afte rsubcutaneous administrati onof either (i) HUMALOG® or (ii) UFAL by using a handheld blood glucose monitor or ELISA on collecte dblood sample s.

Randomization: 5 pigs were used for this study and each pig received each formulati on once. The order in which the formulations were given in was randomized. Blinding: For analysis of pharmacokinetic parameters (t50% up, time to peak, t50% down) pharmacokineti c curves were coded, and were analyzed by a blinded researcher.

Replication. 5 pigs were used in this study and each pig acted as its own control receiving each formulati on(HUMALOG® and UFAL) once.

Streptozotocin induced diabetes in swine : Five femal eYorkshire pigs (Pork Power) were used for our animal studies, which were performed in accordance wit hthe Guidelines for the Care and Use of Laboratory Animals and the Animal Welfare Act Regulations. All protocols were approved by the Stanford Institutional Animal Care and Use Committee Type-l-li. ke diabetes was induced in pigs (25-30 kg) using streptozoto cin (STZ) (MedChemExpress). STZ was infused intravenousl aty a dose of 125 mg/kg and animal swere monitored for 24 hours. Food and administration of 5% dextrose solution was given as needed to prevent hypoglycemia .Diabetes was defined as fasting blood glucose greater than 300 mg/dL.

In vivo pharmacokinetic sand pharmacodynamics in diabetic swine : Five diabetic pigs were faste dfor 4-6 hours. Pigs were injected subcutaneous wilyth a 2-4 U dose of the following formulations: (i) HUMALOG® (100 U/mL, Eli Lilly) or (ii) ultra-fas absorbint g insulin lispro (UFAL) (100 U/mL Zn-free lispro, 2.6 wt.% glycerol, 0.85 wt.% phenoxyethanol, 0.01 wt.% MORPH-NIP23%). Doses were determined based on individual pig insulin sensitivity values wit ha target of a decrease in blood glucose of approximatel y 200 mg/dL. Individu alpigs received the same dose for each treatment group. Pigs received each formulati ononce on separate days and the order of the treatment groups were randomized. Before injection, baseline blood was sampled from an intravenous catheter line and measured using a handhel dglucos emonitor (Bayer Contour Next). After injection, blood was sampled from the intravenous catheter line every 5 minutes for the first 60 minutes, then every 30 minutes up to 4 hours. Blood was collected in K2EDTA plasm atubes (Greiner-BioOne) for analysis wit hELISA. Plasm alispro concentrations were quantified using an Insuli nLispro ELISA kit (Mercodia).

Pharmacokinetic Modelling: The pharmacokinetic model used in this analysis was derived from literature reports. Insuli nconcentrations for injection (linj) ,equilibrium in the interstitium (Lq), and the plasm (Ip)a were numerically solved using a system of differential equations outl, ined below, as a function of time using the SciPy (version 1.2.1) odeint function in Python (version 3.6.8). d^inj ן ן (1) at q = k!* linj ־ k2 * Ieq (2) = k2 * Ieq ־ k3 * Ip (3) Concentrations were initialized such that at t = 0 all insulin was present in linj.

Kinetic rate constants were fit for the normalized pig pharmacokinetic curve sby minimizing the sum of squared errors (SSE) between the generated, normalized insulin plasm aconcentrations derived from the model at the experimental time points from 0 to 90 minutes and the normalized pig plasm ainsulin concentrations for UFAL and HUMALOG®. We assum ethat k! and k3 are species dependent, whil ek\ is both species and formulati ondependent. While minimizing the SSE, we observed that there was no upward bound for £1,uFAL,Pig; such that higher values of £1,uFAL,Pig resulted in increasingly marginall ysmall erSSEs for a given ki and k3. Accordingly, &1,uFAL,p1g was then set at 100,000 min1־. The SSE was minimized by first employing a grid search using SciPy’s optimize brute function and subsequently refining the rate constants by employing SciPy’s optimize minimize function using the L-BFGS-B method. To solve for k\. up al , Human, we assume the following relationship: ki.UFAL.Pig _ ^l.UFAL,Human ^!.Humalog,Pig ^!.Humalog,Human Values for ki, hi !mat ,on®,Human, ki, Human, and fe,Human were used as reported in the literature.

Statistica Analysl is: All resul tsare expressed as a mean ± standard deviation unless specified otherwise FIGs. . 10D-10F are shown as mean ± standard error of the mean. All statistical analyses were performed as general linear models (GLMs) in IMP Pro Version 14. Comparisons between formulations (FIGs. 10D-I and K-M) were conducted using the restricted maximum likelihood (REML) repeated measures mixed model. Suitable transformations applied as needed to meet the assumptions of the methods (z.e. homogeneity of variance, normality of error, and linearity). Time to 50% of peak up, time to peak, and time to 50% peak down were log transformed for analyses to correct for non-homogeneity of variance. Pig was included as a variable in the model as a random effect blocking (control) factor to account for variation in individual pig response.

Statistical significance was considered as p<0.05. For FIGs. 10D-I, post-hoc Bonferroni correction was applied to account for multiple comparisons and significance was adjusted to alpha=0.008.

Results High-throughput screen for insulin stabilizing excipient: The AC/DC excipients prepared as described under Example 1 were evaluated for their potenti alas a stabilizing excipient for insulin using an absorbance-based stresse daging assay, where destabilized insulin aggregates scatter light and increase the absorbance of the solution. Time to aggregation in these assays is defined as a 10% increase in absorbance of the formulation.

Recombinant insuli nwas formulated in PBS at standard formulation concentrations (100 U/mL; 3.4 mg/mL) and tested wit h(i) no polymer excipients, (ii) pluronic L-61 (the mos t similar commerciall avaiy labl polymere both chemicall yand physicall toy Pol oxamer 171 used in INSUMAN® U400), (iii) 1 mg/mL AC/DC excipients, or (iv) 10 mg/mL AC/DC excipients. Recombinant insulin controls, wit hno polymer excipient, aggregated in 13 ± 8 hours in this assay. Formulation wit hpluronic L-61 (1 mg/mL) prolonged aggregation to 27 ± 2 hours, demonstrati ngefficacy of the commercial polymer as an excipient to prevent insulin aggregation. The use of water-soluble carrier homopolyme rexcipient s(1 mg/mL) had no impact on insulin stabilit (FIG.y 5 A), demonstrati ngthat free hydrophili cpolymers are not sufficient to prevent insulin aggregation. This finding is supported by previous work showing that other hydrophilic polymers such as poly(ethylene glycol (PEG)) do not improve insulin stability.

Insuli nstability when formulat edwit hAC/DC excipient swas highly chemistry dependent. Each AC/DC excipient was formulated wit hinsulin and stabilit wasy tested for up to one month (FIGs. 5B-G and Table 3). Formulations comprising AC/DC excipients wit h MPAM and MORPH carrier chemistries demonstrat edthe overall highest improvement of insulin stabilization, especially when combined wit hNIP, TEA and PHE dopants. While many carrier-dopant combinations demonstrated long-term stabilit aty 1 wt.% formulati onconcentrations we, sought to engineer copolymers capable of stabilizing insulin at minimal concentrations in formulation. AC/DC excipients comprising MPAM- PHE, MP AM-TEA, MPAM-TRI, and MORPH-TEA (0.1 wt.%) stabilize insuld in for over 100 hours of stresse daging. These formulations are therefore 7-fold more stabl ethan recombinant insuli nalone and 3-fold more stabl thane those containing pluronic L-61.

Moreover, AC/DC excipients comprising MPAM-NIP, MPAM-AMP, and MORPH-PHE (0.1 wt.% )stabilize insuld in for 30 days of stresse daging, at which point the assay was terminate d.These formulations are 50-fold more stable than insuli nalone, and 24-fold more stable than formulations containing pluronic L-61. These select carriers and dopants are the most hydrophobic amongs tthe monomers screened, suggestin thatg amphiphilic water-soluble copolymer sare most effective at preventing insulin aggregation.

Table 3: Days until aggregation for recombinant insulin formulated wit hAC/DC excipient sat two excipient concentrations (1 mg/mL and 10 mg/mL) Excipient Concentration 1 mg/mL 10 mg/mL Medium High Medium High Dopant Loading Low Low Carrier Dopant DMA NIP 1.61 1.09 1.05 1.21 0.93 2.13 DMA PHE 1.41 0.72 1.13 1.86 2.41 3.91 DMA AMP 0.9 1.49 0.29 1.42 0.79 n.t.

DMA TMA 1.46 1.07 0.15 1.7 n.t. 0.52 DMA TEA 1.13 0.36 0.29 1.5 1.66 1.29 DMA TRI 1.58 0.25 0.25 1.91 1.46 1.09 MORPH NIP 4.39 3.25 3.53 4.79 7.14 3.4 MORPH PHE 3.08 3.85 30 4.82 17.55 30 MORPH AMP 2.09 2.03 1.33 5.15 2.27 1.59 MORPH TMA 0.58 0.37 0.6 0.71 n.t. n.t.

MORPH TEA 4.26 3.62 3.63 21.58 9.25 14.91 MORPH TRI 1.29 0.38 0.22 n.t. n.t. n.t.

HEAM NIP 1.7 1.19 1.79 1.82 3.26 2.59 HEAM PHE 0.59 0.87 1.97 1.87 11.87 30 HEAM AMP 2.22 0.47 0.4 4.8 2.01 2.72 HEAM TMA 12.25 4.99 0.68 3.78 4.01 10.75 HEAM TEA 0.7 0.56 0.87 1.96 28 23.62 HEAM TRI 1.07 0.37 0.17 1.34 22.68 1.34 MP AM NIP 17.98 25.94 30 30 10.93 2.39 MP AM PHE 27.91 7.89 20.37 11.72 3.61 30 MP AM AMP 29.32 30 29.32 30 29.32 28.64 MP AM TMA 22.01 3.51 2.36 21.33 2.35 4.06 MP AM TEA 15.04 9.55 2.63 18.26 6.35 18.25 MP AM TRI 4.56 1.55 0.08 n.t. n.t. n.t.

AM NIP 1.81 1.99 1.7 1.74 1.96 1.76 AM PHE 1.87 1.34 1.43 2.23 3.57 2.24 AM AMP 0.93 0.89 0.87 1.74 3.5 1.76 AM TMA 0.85 0.8 0.63 1.45 2.32 4.43 AM TEA 1.63 1.38 1.49 5.9 3.96 2.49 AM TRI 0.78 0.85 0.81 1.63 3.91 4.06 n.t. indicates not tested.

Stabilization of monomeric insulin with refined screen: Based on the initial recombinant insulin stability screen, copolymers comprising MP AM or MORPH carriers wit hNIP or PHE dopants demonstrated the most promise as candidates for stabilizing monomeric insulin. Previous work by our group demonstrat edthat the equilibrium betwee ninsulin association stat escan be shifted by altering formulation excipients, where a formulati onthat is approximately 70% monomers can be achieved wit hformulati onof zinc-free lispro wit hglycerol and phenoxyethanol. This formulati onfavors the insulin monomer and completel disy sociate thes insulin hexamer. Representative SEC traces of predominantl hexamey ric HUMALOG® and predominantl monomeriy c zinc free UE AL demonstrate the association stat esof insulin in formulati on(FIG. 6). However, insuli n monomers are unstabl ine formulati onand require additiona stl abilizing excipient sto be viable for translation. Further, it will likely be prudent to use the lowest concentration of copolymer excipient possibl eto reduce chronic exposure to the excipient with frequent insulin use typical of diabetes management.

To address this need, a second library of AC/DC excipient swas synthesized to evaluate additiona carril er-dopant ratios wit hour top performing candidate monomers: (i) MP AM and MORPH as carriers, and (ii) NIP and PHE as dopants. Standar dsynthesis practices were implemented to generate this secondary library, which consiste dof copolymer sat DP 50 wit hMORPH or MP AM as carriers and either (i) NIP loaded at 14, 17, 20, 23, or 26 wt.%, or (ii) PHE loaded at 6, 8, 10, 12, or 14 wt.%., respectively, via SEC and 1HNMR spectroscopy (FIG. 7, Table 4).

Table 4. SEC and 1H NMR analysis of polymers synthesized during the second screen targeting DP 50 Carrier M״a Mwa Da wt.% wt.% by NMR Dopant wt.% wt.% by NMR (Target) (Experimental) (Target) (Experimental) MORPH 94 93.68b PHE 6 6.32b 2900 3400 1.17 MORPH 92 91.83b PHE 8 8.17b 3100 3400 1.1 MORPH 90 90.12b PHE 10 9.88b 3100 3400 1.1 MORPH 88 87.93b PHE 12 12.07b 3100 3500 1.13 MORPH 86 85.51b PHE 14 14.49b 3200 3600 1.13 MORPH 86 79.75c NIP 14 20.25c 2900 3300 1.14 MORPH 83 77.9C NIP 17 22.10c 3100 3500 1.13 MORPH 80 77.73c NIP 20 22.27c 3100 3500 1.13 MORPH 77 74.46c NIP 23 25.54c 3200 3800 1.19 MORPH 74 72.23c NIP 26 27.77c 3000 3400 1.13 MP AM 94 93.54d PHE 6 6.46d 4700 5200 1.11 MP AM 92 90.94d PHE 8 9.06d 5000 5400 1.08 MP AM 90 89.05d PHE 10 10.95d 5100 5600 1.1 MP AM 88 87.61d PHE 12 12.39d 4900 5500 1.12 MP AM 86 86.15d PHE 14 13.85d 5000 5500 1.1 MP AM 86 86.33e NIP 14 13.67e 4700 5100 1.09 MP AM 83 82.35e NIP 17 17.65e 4600 5000 1.09 MP AM 80 78.916 NIP 20 21.096 4500 4800 1.07 MP AM 77 77.95e NIP 23 22.056 4400 4800 1.09 MP AM 74 73.11e NIP 26 26.89e 4400 4800 1.09 a Mn (number average molecular weight), Mw (weight average molecular weight), and D (dispersity) determined using size exclusion chromatography calibrate dusing polyethyle neglycol standards. b Weight percentages calculated from post precipitated NMR spectr aof MORPH (5= 3.3-3.7, 8H) and PHE (5= 7.6, 2H). c Weight percentages difficult to determine due to overlapping spectra. Weight percentages estimated from post precipitate NMRd spectra by measuring the more resolved left half of the peak of NIP (5= 4.0, 0.5 H), doubling it, and subtracti ngit from the unresolved peaks of MORPH and NIP(5= 3.2-4.2, 7H (MORPH) 1H (NIP)). d Weight percentages calculated from post precipitated NMR spectr aof MP AM (5= 3.1-3.5, 7H) and PHE (5= 7.6, 2H). e Weight percentages calculated from post precipitate dNMR spectra of MPAM (5= 3.2, 3H) and NIP (5=3.8, 1H).

Using the AC/DC excipient ssynthesized in the second screen, UE AL formulations were prepared wit h0.01 wt.% (0.1 mg/mL) copolymer excipient and insuli naggregation were assessed under stressed conditions using the same assay as the initial screen (Table in Supplement Attal achment A). HUMALOG®, the commercial formulati onof insulin lispro, aggregated under these conditions within 6 hours. UFAL without AC/DC excipients aggregated in 1.3 ± 0.3 hours, demonstrating the severe instabilit ofy the insulin monomer in solution. All UFAL formulations stabilize witd hMORPH-PHE or MPAM-PHE AC/DC excipient sexhibited stabilit iesto stresse daging at least equivalent to commercial HUMALOG®. Copolymer scomprising MP AM with 14 wt.% PHE (MPAM-PHE14%) and MORPH with 12 wt.% PHE (MORPH-PHE12%) were among the top candidates, extending UFAL formulati onstabilit toy 27 ± 2 hours and 25 ± 5 hours, respectivel (Figs.y 8B-C).

MPAM-NIP copolymer sdemonstrated limited efficacy in stabilizing the monomeric insulin; however, MORPH-NIP copolymers extended monomeric insulin stabili ty compared to HUMALOG®. Indeed, copolymers comprising MORPH wit h23 wt.% NIP (MORPH-NIP23%) extended UFAL formulati onstability to over 25 ± 1 hours. The top candidate AC/DC excipient safter the second screen were MPAM-PHE14%, MORPH- PHE12%, and MORPH-NIP23%. While these copolymers demonstrated high efficacy, MPAM-PHE14% and MORPH-PHE12%also demonstrate decred ased solubilit andy LCST- like phase separation behavior at physiological temperature when present at higher concentrations. Thus, MORPH-NIP23% was chosen as the top candidate used to stabilize our UFAL formulati onin subsequent in vivo studies. In vitro and in vivo bioactivity assays were used to corroborate the transmittance data and confirm UFAL integrit ybefore and after aging. UFAL showed no loss in activity after 12 h of stresse daging in either the cellular assay for phosphorylation of Ser473 on AKT or in diabetic rats lowerin gblood glucose levels (FIG. 9).

To verify that formulati onwit hMORPH-NIP23% did not alter the lispro association state equilibrium away from the monomer form, NMR DOSY was used (FIG. 8D). NMR DOSY indicate dthe diffusion rate of lispro under formulation conditions (100 U/mL lispro, 2.6 wt.% glycerol, 0.85 wt.% phenoxyethanol, and 0.1 wt.% MORPH-NIP23%) had a diffusion rate of 2.0 x 1010־ m2 s1־, corresponding to a hydrodynami cradius of 1.2 nm, which corresponds to reported literature values of the insulin monomer (28). NMR DOSY also provided insight into the stabilizatio mecn hanism of the polymer excipients. MORPH- NIP23% diffused at a slower rate than insulin, suggesting that the mechanism of stabilizatio isn not related to excipient-insuli complexatn ion and co-diffusion. These data support the hypothesis that copolymer-interface interactions are the primary mechanism driving monomeric insulin stabilizatio inn formulation.

Pharmacokinetics and pharmacodynamics of UFAL formulati onin diabetic swine: To assess the ultra-fas potentialt of the monomeric insulin formulations, pharmacokineti c studi eswere conducted in a swine model of insulin-deficient diabetes. Faste ddiabetic swine were treated wit heither (i) commercial HUMALOG® or (ii) UFAL (100 U/mL lispro, 2.6 wt.% glycerol, 0.85 wt.% phenoxyethanol, and 0.1 wt.% MORPH-NIP23%) at a dose of 2-4 U of insulin lispro, depending on the insulin sensitivi tyof each pig. Pigs had a starting blood glucose level between 330-430 mg/dL and insuli ndoses were chosen to reduce blood glucose to approximatel 100y mg/dL. The insulin dose given to each pig was consistent between treatment groups and blood glucos edepletion was similar in both HUMALOG® and UFAL treatments (FIG. 10A and FIG. 11). Plasm aconcentrations of lispro were measured over time by enzyme-linked immunosorbent assay (ELISA) to assess pharmacokinetics following subcutaneous injection of each of the treatment groups .No difference in overall exposure (AUC210) between groups was observed (FIG. 10C). Percent exposure at various time points was analyzed by looking at the AUC1/AUC210. This analysis shows increased exposure for UFAL compared to HUMALOG® at 10 min and 20 min timepoints (FIGs. 10D-10I). Mean residence time (MRT) is commonl yreported for formulati onpharmacokinetics. MRT is commonly described as area under the moment curve (AUMC) divide dby area under the curve (AUC) for intravenous injections; however, when drugs are administere dsubcutaneously the mean absorption time (MAT) must also be considered. When there is an absorption phase, AUMC/AUC = MAT + MRT.

The ratio of the area under the moment curve (AUMC) to the area under the curve for the pharmacokineti cplot (AUMC/AUC) was calculated and plotted, showing no difference betwee nUFAL and HUMALOG® treatment (FIG. 13). This was not surprising, as we woul dexpect the clearance rate from the blood to be similar for both HUMALOG® and UFAL (both are insulin lispro) and the magnitude of MAT in comparison to MRT woul d be smal l,thereby masking differences between formulations.

Alternatively exposure, metric sare commonly reported for fast-acting insuli n formulations to describe the formulati onpharmacokinetics. The "time-to-onset" rate of fast-acting insulins is often determined using two metrics: (i) time-to-50% of the normalized peak height on the way up following administrati on(denoted "50%-up"), and (ii) time-to-peak insulin plasm aconcentration. Normalized plasm aconcentration measurements were used to compare the time-to-peak lispro concentrations between commercial HUMALOG® and UFAL treatment groups (FIGs. 10J-10M). Pigs exhibited almos t2-fold faster HUMALOG® pharmacokinetic scompared to humans . UFAL demonstrated faster absorption than HUMALOG®, whereby UFAL time to 50% of peak up (5 2 ؛ min) was 2.4-fold faster than HUMALOG® (12 ± 6 min), and UFAL time to peak (9 ±4 min) was 2.8-fold faster than HUMALOG® (25 ± 10 min). The exposure duration, defined as the time to 50% of the normalized peak height on the way down following peak exposure concentrations (time to 50% peak down), for UFAL (28 ± 8 min) was 1.9-fold shorter than for HUMALOG® (54 ± 21 min).

Modeling UFAL pharmacokinetics in humans: To better understand how the fast onset and short duration demonstrated by UFAL in pigs woul dtranslat toe humans ,a pharmacokineti cmodel was adapted from Wong et al. (J. Diabetes Set. Technol. (2008) 2:658-671) to approximate UFAL pharmacokinetic sin humans. The model was construct edsuch that rapid-acting insulin analogues (HUMALOG®) injected into the subcutaneous space (linj) dissociate and diffuse wit ha rate constant, k\, into the interstitium (leq), absorb wit ha rate constant, ^2,into the plasma (Ip), and are subsequent lycleared by severa lmechanisms that can nonetheless be approximated by a single elimination constant, fa (FIG. 12A). It was assumed that fa and fa were species dependent, fa was formulati on and species dependent, and the ratio of fa between formulations was species independent.

Because the UFAL formulati onwas composed of insuli nmonomers and dimers, the tim e necessary to reach equilibrium in the interstitium was expected to be appreciably lower than for HUMALOG®. Indeed, when fitting the experimental pig pharmacokinetic sfor subcutaneous administration of UFAL, k\ trended towards infinity, meaning that UFAL effectively bypassed the first model compartment and the insulin monomers reached equilibrium in the subcutaneous space immediatel (Tably e 6). The fits for the pig pharmacokineti cdata for both UFAL and HUMALOG® are presented in FIG. 12B, and a comparison betwee n the model predictions and experimental data wit h relevant pharmacokineti cmetrics are presented in FIG. 14, Supplemental Attachment A. The infinitely large k\ determined for UFAL in pigs was translat edto a human pharmacokineti c model and used to estimate UFAL pharmacokinetic swhile maintaining k! and fa values reported in the literature (FIG. 12C).

Table 6. Rate constants used for modeling PK curve sin Example 2. insuli nvariant U(min-1) O (min1־) k3 (min1־) Species HUMALOG® 0.091 0.042 0.27 Pig UFAL IR) 0.042 0.27 Pig human HUMALOG® 0.0104" 0.0604" 0.16a human UFAL co 0.06042 0.16a 3Human pharmacokinetic rate constants from Wong etal., J. Diabetes Sci. Technol. (2008) 2:658- 671.

The model predict shuman UFAL time to onset (i.e., 50%-up) of 2.5 minute s,peak exposure at 10 minutes, and duration of exposure (i.e., 50%-down) of 28 minutes (FIG. 12D). In comparison, using parameters reported in the literature, the model predict "srapid- acting" insulin analogues (RAI), such as HUMALOG®, to exhibit a time to onset of 14 minutes peak, exposure at 43 minutes and, a duration of exposure of 157 minutes (FIG. 12C). While the RAI model underestimates the time to onset of exposure (t50% up), the predicted curve robustly captures publishe dclinical pharmacokinetic data for peak and duration of HUMALOG® exposure. The pharmacokinetic modeling, therefore ,predict s UFAL to exhibit kinetics that are more than 4-fold faster than current "rapid-acting" insulin formulations Further. comparison to clinical data for rapid-acting insulin formulations demonstrate thats UFAL is predicted to be faster than even second generation rapid-acting insulin formulations such as Fiasp (Novo-Nordisk) and BioChaperone Lispro (Adocia) (FIG. 12E).

In diabetic swine ,this UFAL formulati onexhibited ultra-fas pharmt acokinetics, wit happroximately two-fold faster time to onset and two-fol shorted rduration of exposure than HUMALOG®, a commercial "rapid-acting" insuli nformulati onusing the same insulin molecule Lispro. These results suggested that this UFAL formulati onmore closel y mimics endogenous insulin secretion in healthy individual ands highlighted that this formulati onis promising for enhancing diabetes management .Even the incremental improvement in pharmacokinetics over current "fast-acting" insulin formulations observed for Fiasp, a faster-acting version of NOVOLOG® (commercial aspart formulation), have shown numerous clinical benefits. While Fiasp shows a modest 10 minute reductio inn time to peak action and 15 minute reduction in duration of action over "rapid-acting" insulin formulations, Fiasp use nevertheless reduced post-prandi alglucose excursions and reduced HbAlc levels in patients wit hdiabetes .In contrast in, diabetic pigs, where the observed insuli npharmacokinetics were twice as fast as in humans ,UFAL reduced time to peak exposure by 16 minutes and reduced duration of exposure by 26 minutes compared to HUMALOG®. The resul tsobserved in diabetic pigs, combined wit h the model predicted human UFAL pharmacokinetics suggested that UFAL may have absorption kinetics that are unprecedented in an injectable insulin formulation. If realized in human clinical studies, these kinetics woul dbe approaching the ultra-fas kinett ics of AFREZZA®, the commerciall avaiy labl inhale able insulin. However, unlike AFREZZA®, UFAL is an injectable formulation, which enables more accurat edosing regimens and compatibilit wity hpump and closed-loop systems, providing UFAL the potenti alto improve post-prandial glycemic control in patients wit hdiabetes.

Taken together these studi esidentified a copolymer excipient for protein stabilizatio andn show its utility in stabilizing an ultra-fast absorbing insulin formulation.

Initial cytotoxicit experiy ment ssuggested that the copolymer excipient is not toxic at doses an order of magnitude higher than those used in insulin formulations (FIG. 13). Initial biocompatibilit studiy eswith UFAL in diabetic rats also corroborated the cytotoxicity results, indicating that UFAL should be wel ltolerated (FIG. 14).

Example 3: Ultra-fast insulin-pramlintide co-formulation Materials: An amphiphili c acrylamide copolymer excipient acryloylmorpholine77%-N-isopropylacrylamide23 (M0N% i23%) was prepared according to the procedure sas set forth above under Example 1, UFAL. HUMALOG® (Eli Lilly) and pramlintide (BioTang) were purchased and used as received .For zinc-free lispro, Zinc(II) was removed from the insulin lispro through competitive binding by addition of ethylenediaminetetraacet acidic (EDTA), which exhibits a dissociation binding constant approaching attomolar concentrations (Kd~1018־ M). EDTA was added to formulations (4 eq with respect to zinc) to sequest erzinc from the formulati onand then lispro was isolated using PD MidiTrap G-10 gravity columns (GE Healthcare) to buffer exchange into water.

The soluti onwas then concentrated using Amino Ultra 3K centrifugal units (Millipore ) and reformulat edwit h2.6 wt.% glycerol, 0.85 wt.% phenoxyethanol in 10 mM phosphate buffer (pH=7.4). All other reagents were purchased from Sigma-Aldrich unless otherwis e specified.

In-line Size Exclusion Chromatography Multi-Angel Light Scattering (SEC- MALS): Insuli nassociation state composition for monomeric insulin formulati onwas obtained using SEC-MALS as previousl reportedy (Maikawa et al., Adv. Ther. (2019) 75:1900094). Zinc-free insulin lispro was evaluated in a buffer containing glycerol (2.6%) and phenoxyethanol (0.85%). Briefly, number-averaged molecul arweight (MW) and dispersit y(D = Mw/Mn) of formulations were obtained using size exclusion chromatography (SEC) carried out using a Dionex Ultimate 3000 instrument (including pump, autosampl er,and colum ncompartment) outfitt edwit ha Dawn Heleos II Mult i Angle Light Scattering detector, and a Optilab rEX refractive index detector. The column was a Superose 6 Increase 10/300 GL from GE healthcare. Data was analyzed using Astra 6.0 software. The fraction of each insulin association state was derived by fitting the experimentall yderived number-average and weight-average molecula weigr hts to Equation 1 and Equation 2 below, m. d and h, respectively repre, sent the molar fractions of monomeric, dimeric and hexameric insulin while I represents the molecul weiar ght of monomeric insulin lispro. The solve wasr constrained so that m^dvh=\ while m. d and h remain between 0 and 1.

Mn = m*I + d*2I + h*6I (1) ,, m*Z2+d*4Z2+Z1*36Z2 Mw =---------------------- (2) w m*I+d*2I+h*6I v 7 In vitro stability evaluati onof insuli nand pramlintide: Aggregation assays used to evaluat ste abilit wery e adapted from Webber et al. (Proc. Natl. Acad. Set. U.S.A. (2016) 113:14189-14194). Briefly, formulations were aliquote 150d pL per well (n = 3/group) in a clear 96-wel platel and sealed wit hoptically clear and thermally stable seal (VWR). The plat ewas incubated in a microplate reader (BioTek SynergyHl microplat readee r) at 37 °C with continuous agitation (567 cpm). Absorbance readings were taken every 10 minutes at 540 nm for the duration of the experiment. The formation of insulin or pramlintide aggregates leads to light scattering and a reductio nin the transmittanc ofe samples (time to aggregation = time to 10% change in transmittance) Control. sincluded: (i) HUMALOG® (100 U/mL), (ii) HUMALOG® (100 U/mL) + Pramlintide (1:6 lispro:pramlintide), (iii) zinc-free lispro (100 U/mL lispro, 2.6 wt.% glycerol, 0.85 wt.% phenoxyethanol, pH=7.4). The stabilit ofy an insulin-pramlintide co-formulation (100 U/mL lispro, 1:6 lispro:pramlintide 2.6, wt.% glycerol, 0.85 wt.% phenoxyethanol , pH=7.4) mixed with 0.1 mg/mL M0Ni23% was evaluated.

Streptozotocin induced model of diabetes in rats: Male Sprague Dawley rats (Charles River) were used for experiments. Animal studi eswere performed in accordance wit hthe guidelines for the care and use of laboratory animals; all protocols were approved by the Stanford Institutional Animal Care and Use Committee The. protocol used for streptozoto cin(STZ) induction adapted from the protocol by Wu and Huan. Briefly ,male Sprague Dawley rats 160-230 g (8-10 weeks) were weighed and fasted in the morning 6- 8 hours prior to treatment wit hSTZ. STZ was diluted to lOmg/mL in the sodium citrate buffer immediatel beforey injection. STZ solution was injected intraperitoneall aty 65mg/kg into each rat. Rats were provided with water containing 10% sucros efor 24 hours after injection wit hSTZ. Rat blood glucose levels were tested for hyperglycemi adail y after the STZ treatment via tail vein blood collection using a handheld Bayer Contour Next glucose monitor (Bayer). Diabetes was defined as having 3 consecutive blood glucose measurements >400 mg/dL in non-fasted rats.

In vivo pharmacokinetic sand pharmacodynamics in diabetic rats: Diabetic rats were fasted for 4-6 hours before injection. For pharmacokineti cexperiment srats were injected wit h1U insulin formulati on(~2U/kg) followe immd ediately (< 30 seconds after injection) by oral gavage wit hIg/kg glucos esolution. Formulations tested were: (i) HUMALOG®, (ii) separate injections of HUMALOG® and pramlintide (1:6 pramlintide:lispro, pH=4), (iii) insulin-pramlintide co-formulation (100 U/mL lispro, 1:6 lispro:pramlintide 2.6, wt.% glycerol, 0.85 wt.% phenoxyethanol, 0.1 mg/mL M0Ni23%, pH=7.4). A cohort of 11 rats each received each formulati ononce, and the order the formulations were given in was randomized. To allow for accurate dosing and to avoid dilution effects (dilutio favorsn the insulin monomer) formulations were diluted two-fold (10 pL formulati on+ 10 pL formulati onbuffer) immediatel beforey administration. After injection, blood glucose measurements were taken using a handheld glucose monitor (Bayer Contour Next) and additiona lblood was collected (Sarstedt serum tubes) for analysis wit hELISA. Timepoints were taken every 3 minutes for the first 30 minutes then, every 5 minutes for the next 30 minutes then, at 75, 90, and 120 minute s.Serum pramlintide concentrations were quantified using a human amylin ELISA kit (Millipore Sigma). Serum lispro concentrations were quantified using Northern Lights Mercodia Lispro NL-ELISA. A second pharmacodynamics experiment was performed to try to better match insulin dose with oral glucose dose to better simulat meale -time glucos e management .The same formulations were tested but doses were changed to 0.75 U/kg insulin delivered subcutaneous imlymediatel beforey oral gavage with 2 g/kg glucose. A pL Hamilton syringe was used to allow accurat edosing of undiluted (100 U/mL) formulations. A cohort of 10 rats each received each formulati ononce, and the order the formulations were given in was randomized. Only glucos ewas measured and timepoints were taken every 5 minutes for the first hour, followe byd measurements at 75, 90 and 120 minutes.

Gastric emptying in diabetic rats: Acetaminophen was used as a model compound to evaluate gastri cemptying at mealtimes Diabet. ic rats were fasted for 4-6 hours before experiment start Rat. s were then injected subcutaneously wit hone of the following formulations (2 U/kg): (i) HUMALOG®, (ii) separate injections of HUMALOG® and pramlintide (1:6 pramlintide:lispro, pH=4), (iii) insulin-pramlintide co-formulation (100 U/mL lispro, 1:6 lispro:pramlintide, 2.6 wt.% glycerol, 0.85 wt.% phenoxyethanol ,0.1 mg/mL M0Ni23%, pH=7.4). To allow for accurate dosing and to avoid dilution effects (dilutio favorsn the insuli nmonomer) formulations were dilut edtwo-fol d(10 pL formulati on+ 10 pL formulati onbuffer) immediatel beforey administration. A cohort of 11 rats each received each formulati ononce, and the order the formulations were given in was randomized. Acetaminophen was administere dvia oral gavage as a slurr yin phosphate buffer (100 mg/kg) immediately after insulin administration. (Tips of feeding tubes were dipped in glucos esolution before oral gavage to reduc e stress of administration). Blood samples were collecte ford ELISA (Neogen) at -30, 0, 15, 30, 60, 90, 120, ands 150 minutes after injection.

Statisti cs:All resul tsare expressed as mean ± standard error (SE) unless specified otherwise. All statistical analyses were performed as general linear model sin IMP Pro version 14. Comparisons between formulations were conducted using the restricte d maximum likelihood repeated measures mixed model. Post-hoc Tukey HSD test fors multiple comparisons was applied when formulati onwas a significant fixed effect ,and adjust edp-value weres reported. Rat was included as a variable in the model as a random effect blocking (control) factor to account for individual variation in rat responses. (Each rat received every formulati onand acted as its own control). Statistical significance was considered as P < 0.05. For FIGs. 18H-18L, post-hoc Bonferroni correction was applied to account for comparison of formulations at multiple exposure timepoints (In addition to Tukey HSD correction) and significance was adjust edto a=0.01.

Results Stabilization of an insulin-pramlintide co-formulation: Characterization of M0Ni23% molecul arweight and monomer composition can be found in Table 7. Resul ts shown in Example 2 demonstrated the utility of M0Ni23% as a stabilizing excipient for monomeric insulin. The propensit yof insulin and pramlintide to aggregate to form amyloid fibrils, which are primarily initiate dat hydrophobic interfaces, makes them strong candidates for stabilizatio usingn M0Ni23%. Therefore, it was hypothesized that the same M0Ni23% may also be used to physically stabilize an ultrafas meat ltim insule in-pramlintide co-formulation to enable a singl eformulati onwit hincreased pharmacokinetic overlap betwee nthese two hormones.

Table 7: MoNi copolymer excipient characterization.

Carrier Monomer wt% wt% by Dopant Monomer wt% wt% by M״a M״a Da NMR (Tar NMR (Target) (Exp) get) (Exp) 77 23 3200 3800 1.19 Acryloylmorpholine 74.5 b N-isopropylacrylamide 25.5 b (Mo) (Ni) a Determined using Size Exclusion Chromatography calibrated using polyethyle neglycol samples. b Weight percentages difficult to determine due to overlapping spectra. Weight percentages estimat edfrom post- precipitated NMR spectra by measurin gthe more resolved left half of the peak of N-isopropylacrylami de(5= 4.0, 0.5 H), doubling it, and subtracti ngit from the unresolved peaks of Mo and Ni (5= 3.2-4.2, 7H (Mo) 1H (Ni)).

Zinc-free lispro in the presence of glycerol (2.6 wt.%) and phenoxyethanol (0.85 wt.%) as tonicit yand antimicrobial agents ,results in a formulati onwith a high monomer content. This resul wast confirmed in the current study where 83% monomers, 17% dimers and 0% hexamers in formulati onwas observed as analyzed by SEC-MALS (FIG. 15E and FIG. 16). In comparison, commercial HUMALOG® is >99% hexameric. For SEC-MALS measurements insuli, nassociation stat ewas tested alone wit honly smal lmolecul e excipient sbecause both pramlintide and the M0Ni23% excipient are of similar molecula r weight and woul dprevent the calculation of monomer content in formulati onby SEC- MALS. The addition of M0Ni23% has been shown not to alter the insulin association stat e by diffusion-ordered nuclea magnetr ic resonance spectroscopy (DOSY-NMR). It was not anticipated that the presence of pramlintide woul dalter the insulin association state.

The insulin-pramlintide co-formulation was composed of zinc-free lispro (100 U/mL), pramlintide (1:6 molar ratio pramlintide:lispro), glycerol (2.6 wt.%), phenoxyethanol (0.85 wt.%), and M0Ni23% (0.1 mg/mL) in phosphate buffer at pH~7 (FIG. 15D). A pramlintide ratio of 1:6 was chosen in order to compare wit hprevious work using the CB[7]-PEG stabilize insuld in-pramlintide co-formulation in diabetic pigs. Further, a ratio of 1:6 is similar to high endogenous insulin-pramlintide ratios reported in the literature as well as within the range of ratios indicate dto be most effective by in silico experiments. Formulatio stabiln ity was assessed using a stressed aging assay. As insulin and/or pramlintide aggregates form, they scatter light which can be measured by absorbance. As defined herein, formulati onaggregation is defined as a 10% or greater change in transmittance. The co-formulation tested was shown to be stable for 16.2 ±0.1 hours, twice as long as commercial HUMALOG® which aggregated after 8.2 ± 0.5 hours (FIG. 15F). The direct additio nof pramlintide to HUMALOG® resulted in a translucent formulati onimmediately upon mixing which had 5-25% reduced transmittanc compae red to HUMALOG® alone (FIG. 17). This mixture reached the aggregation threshold afte r8 ± 3 hours, which was highly variable due to the variable initial transmittanc Zince. -free lispro alone was mostly monomeric and highly unstable, aggregating rapidly after 5.7 ± 0.1 hours.

Pharmacokinetics and pharmacodynamics in diabetic rats: After establishi ngthe stabilit ofy the insulin-pramlintide co-formulation, the pharmacokinetic swere evaluated in vivo to determine if the use of monomeric insulin result edin increased pharmacokineti c overlap for insulin and pramlintide. The co-formulation was tested against control sof HUMALOG® alone and separate injections of insulin and pramlintide (FIG. 18). A high dose of each formulati on(2 U/kg) was given to each rat followe byd oral gavage wit h glucose solution (1 g/kg). A simila rmagnitude in glucose depletion was observed in all three formulations however, glucose depletion for the co-formulati onhad a trend for more rapid action (faste rglucos edepletion) and shorter duration of action (faste rglucose recovery) compared to HUMALOG® and separate injection controls (FIG. 18A). This trend was mirrored in the insulin lispro pharmacokinetics where, a trend for faster onset and time to peak exposure was observed for the co-formulation (FIGs. 19A-19C). There was a difference in duration of action, defined as 50% of peak down, betwee nformulations (F2, 20=7.07, P=0.0048). The co-formulation had shorte rduration of action (22 ± 2 minutes) com-pared to separate injections (34 ± 3 minute s,P=0.0034), and a trend for shorte rduration of action compared to HUMALOG® (27 ± 2 minute s,P=0.24) (FIGs. 19A- 19D). Faster onset was also corroborated using exposure ratios - the fraction of the area under the curve (AUG) at a given time point over the total (AUC1/AUC120). The co- formulati onshowed a greater fraction of total exposure compared to HUMALOG® and separate injections at 6-, 15- and 30-minut etimepoints (FIGs. 19E-19I). There was no difference in insulin lispro or pramlintide area under the exposure curve between formulations (FIG. 20). As expected, there were no differences observed between pramlintide kinetics delivered as separate injections versus in the co-formulation (FIGs. 19J-19M, FIG. 21).

The shift of the co-formulation insulin lispro pharmacokinetic curve to the left was confirmed by overlaying the insulin pramlintide curves for delivery by separate injections or co-formulation and comparing overlap time (FIG. 22). Overlap was defined as the ratio of overla pover total peak widt hat half peak height (overla p= (lispro + pramlintide - overlap). As hypothesized, delivery of monomeric insulin wit hpramlintide in a co- formulati onresulted in increased overlap (0.75 ± 0.06) compared to separate injections (0.47 ± 0.07, Fl, 10=6.96, P=0.025) (FIG. 10C). The faster insuli nkinetics and increased overlap betwee ninsulin and pramlintide observed in our co-formulation more closel y mimic insulin-pramlintide secretion at mealtimes.

Gastric emptying of acetaminophen in diabetic rats: Once the faster insuli n kinetics and increased overlap betwee ninsulin and pramlintide were observed for the co- formulati onof the present disclosure, the next step was to determine if there were mealtime benefits to this co-formulation compared to standard administrations of HUMALOG® alone or HUMALOG® and pramlintide administere separad tel y.Acetaminophen was used as model cargo to confirm pramlintide function by testing its ability to dela ygastri c emptying after formulati onadministration (FIG. 23). It was expected that pramlintide in both separate administrations and in the co-formulation woul dresul int delayed gastri c emptying compared to HUMALOG® alone. Indeed, the time to peak acetaminophen concentration was delayed until 76 ± 5 minutes for separate injections and 68 ± 6 minutes for the co-formulation compared to 35 ± 5 minutes for HUMALOG® alone, demonstrating there was no difference in time to peak acetaminophen betwee nseparate injections and the co-formulation (FIG. 23C).

Mealtim eglucose challenge in diabetic rats: The co-formulati onwas further tested in a simulated mealtim challe enge wit ha low dose of subcutaneous insulin (0.75 U/kg) and a high dose of glucose (2 g/kg) administere dby oral gavage (FIG. 24). In contras tto the glucose measurements in the pharmacokineti cexperiment swhere insuli n was dominant, this experiment aimed to reduce the insulin dose and increase the glucose load to better simulat meae ltim glucosee management. All three formulations had similar contro lof the glucose peak (FIG. 24C). However, rapid insulin onset combined wit h delayed gastri cemptying and short duration of action was observed for the instant co- formulati onof insuli nand pramlintide wit hM0Ni23% copolymer, resulting in tighte r contro lof this mealtim eglucose spike while also reducing the magnitude of glucose depletion below baseline level (FIG.s 24B and FIG. 24D). In contrast while, the delayed gastric emptying for the separate injection formulations resulted in rapid glucose depletion and contro lof the mealtim glucosee spike, it also result edin greater glucos edepletion below baseline. The HUMALOG®-only administrati onresulted in a similar glucose curve to separate administrations of insulin and pramlintide but wit hdelayed glucose depletion since glucos release ewas not delayed. These resul tssuggested that the co-formulation may enable good prandial glucose control while also reducing post-prandial hypoglycemia.

This study showed that a co-formulation of monomeric insulin lispro and pramlintide had ultrafas kinetit cs wit ha high degree of overlap, which resulted in improved glucose management after a glucose challenge. This formulati onused amphiphilic acrylamide copolymer excipient M0Ni23% as a stabilizing agent and was physically stable twice as long as commercial HUMALOG® in a stresse agind g assay. The pramlintide in the co-formulati onresult edin delayed gastric emptying similar to separatel y administered pramlintide. Further, the combined effects of ultrafas insulint and pramlintide delivery synchronized in the co-formulation result edin reduced glucose depletion below baseline measurements while maintaining control of the initial glucos e spike in the simulated "mealtim" eglucose challenge. These results suggest that the co- formulati onhas potential to improve glucose management by reducing the risk of post- prandial hypoglycemia, while reducing patient burden.

The data in rats showed only trend sfor increased time to onset (50% of peak up) and time to peak for lispro in the co-formulation compared to HUMALOG® and separate injections. However, AUG ratios representing the fraction of exposure at various timepoints showed that the co-formulation had a greater fraction of early lispro exposure than separate injections and HUMALOG® up until 30 minutes afte rinjection. These observations were surprising, because this study was performed in diabetic rats that have much faster insuli nabsorption rates on account of their loose skin that resul int a larger surface area for subcutaneous absorption compared to humans (FIGs. 25A-25C). Indeed, studi escomparing rapid-acting insulin analogues and recombinant human insulin, which have distinct differences in time to onset ,do not observe differences when compared in rats. A previous study of monomeric lispro in diabetic pigs showed that time to onset and time to peak were twice as fast for monomeric lispro compared to HUMALOG®. Further, comparison of HUMALOG®, monomeric lispro, and pramlintide kinetics between rats and pigs corroborated previous modeling, suggesting these ultrafas kinetit cs will be conserved across species (to humans) (FIGs. 26A-26C and FIGs. 27A-27C). Where HUMALOG® time to peak almost doubled from rats (13 ± 1 minutes) to pigs (25 ± 4 minutes), time to peak for monomeric lispro (delivered as part of the co-formulation in rats) was similar in both species (11 ± 1 minutes in rats and 9 ± 2 minutes in pigs) (FIG. 26). The conservation of time to peak exposure from rats to pigs was highly promising for the translation of these ultrafas insult in kinetics to human trials and woul dresul int kinetics faster than current commercial formulations (FIG. 26).

Example 4: Copolymer sas "drop-in" excipients for insulin formulations Materials: HUMULIN® R (Eli Lilly) was purchased and used as received .Solvents N,N-dimethylformamide (DMF; HPLC Grade, Alfa Aesar, >99.7%), hexanes (Fisher, Certified ACS, >99.9%), ether (Sigma, Certified ACS, Anhydrous, >99%) and CDCI (Acros, >99.8%) were used as received. Monomers N-(3-methoxypropyl)acrylam ide (MPAM; Sigma, 95%), 4-acryloylmorpholine (MORPH; Sigma, >97%) were filtered wit h basic alumina prior to use. Monomers N-phenyl acrylamide (PHE; Sigma, 99%) and N- isopropyl acrylamide (NIP AM; Sigma, >99%) were used as received .RAFT chain transfer agents 2-cyano-2-propyl dodecyl trithiocarbonate (2-CPDT; Strem Chemicals, >97%) and 4-((((2-carboxyethyl)thio)carbonothioyl)thio)-4-cyanopent anoicacid (BM1433; Boron Molecular, >95%) were used as received .Initiator 2,2’-azobis(2-methyl-propionitril e) (AIBN; Sigma, >98%) was recrystallized from methanol (MeOH; Fisher, HPLC Grade, >99.9%) and dried under vacuum before use. Z-group removing agents lauroyl peroxide (LPO; Sigma, 97%) and hydrogen peroxide (H2O2; Sigma, 30%) were used as received .

Streptozotocin (99.58%) was purchased from MedChem Express. All other reagents were purchased from Sigma-Aldrich unless otherwise specified.

Surface Tension: Time resolved surface tension of the air-soluti oninterface was measured wit ha Platinum/Iridium Wilhelmy plate connected to an electrobalanc (KSVe Nima, Finland). The Wilhelmy plat wase partiall immy ersed in the aqueous solution in a Petri dish, and the surface tension of the interface was recorded for 50 minutes from the formation of a fresh interface. Equilibrium surface tension values (t = 50 min) were reported as these values more closely describe the environment in a stored vial before agitation. Two replicate weres taken and averaged.

Interfacial Rheology: Interfacial shear rheology was measured using the Discovery HR-3 rheometer (TA Instruments) wit han interfacial geometry comprising of a Du Nouy ring made of Platinum/Iridium wire (CSC Scientific, Fairfax, VA, catalog No. 70542000).

Before each experiment, the Du Nouy ring was rinsed wit hethanol and water and flame treated to remove organic contaminants The. soluti onchamber consiste dof a double-wall Couett flowe cell wit han internal Teflon cylinder and an external glass beaker. A time- sweep was performed with a strain of 1% (withi nthe linear regime) and a frequency of 0.05 Hz (low enough for instrument inertia to not be significant). Interfacial complex shear viscosity was measured for 30 minute s.The experiment was repeated in triplicate.

Polyme r Synthesis: Polymers were synthesized via reversible addition fragmentation transfer as described in Example 1 above. The procedure to synthesize M0Ni23% AC/DC excipient is as follows and was nearly identical for all other carrier/dopant combinations ,where only the carrier/dopant selection and concentrations were changed. MORPH (566 mg, 4.02 mmol ,36.5 eq.), NIPAM(168mg, 1.485 mmol, 13.5 eq.), 2CPDT (38 mg, 0.11 mmol ,1 eq.) and AIBN (3.6 mg, 0.02 mmol ,0.2 eq.) were combined and diluted wit hDMF to a total volume of 2.25 mL (33.3 w/v vinyl monomer concentration) in an 8 mL scintillat ionvial equipped wit ha PTFE septa. The reaction mixture was sparged wit hnitrogen gas for 10 minutes and then heated for 12 hours at 65 °C. To remove the Z-terminus of the resulting copolymer, AIBN (360 mg, 2.2 mmol ,20 eq.) and LPO (88 mg, 0.22 mmol, 2 eq.) were added to the reaction mixture, which was then sparged wit hnitrogen gas for 10 minutes and heated for 12 hours at 90 °C. Z-group removal was confirmed by the ratio of the refractive index to UV (=310 nm) intensity in size exclusion chromatography (SEC) analysis. Resulting copolymers were precipitated three times from ether and dried under vacuum overnight.

In Vitro Insuli nStabilit Assayy (Accelerate dAging): 50 pL of AC/DC excipient (M0Ni23%, MpPhe8%, M0Phe6%) in milli-Q water (2.1, 21, or 105 mg/mL) or 50 pL of milli- Qwater was added to 1 mL of HUMULIN® R (Eli Lilly 100 U) in a glass autosampler vial (J.G. Finneran, 2.0 mL Clea rRAM.™ Large Opening Vial, 12 x 32mm, 9mm Thread) and capped, yieldin g95U HUMULIN® either as a control or formulated wit h0.01, 0.1, or 0.5 wt.% AC/DC excipient .These vial swere incubated at 37 °C and agitated at 150 RPM for 2, 4, and 6 months (in addition, HUMULIN® only control was agitated at 2 weeks and 1 month). The preparation of formulations was staggered so that all samples reached their endpoint age at the same time. Vials were refrigerated until testing upon reaching selected aging timepoint. Following initial transmittance experiments, all furthe r experiment swere done with formulations wit h0.01 wt.% AC/DC excipient to minimize copolymer concentration. In addition, 500 pL of 2.1 mg/mL M0Ni23% or milli-Q water were added to 10 mL of unadultera tedHUMULIN® (Eli Lilly 100 U) in its commercial vial to generate 95U HUMULIN® control and formulation wit h0.01 wt% AC/DC excipient. These vial swere placed in the original individual packaging boxes wit hthe instruction papers. These packages were incubated at either 37 °C or 50 °C until significant opacity change. 300-400 pL aliquots were removed every 24 hours for the first 7 days and refrigerated. Following that inter, mittent aliquot weres taken to conserve volum e.Every 24 hours, the bottoms of the vial swere photographed to track the change in opacity.

Methods for aggregation assays for recombinant human insulin were adapted from Webber et al. Formulation samples were plated at 150 pL per wel lin a clear 96-wel lplat eand an absorbance reading was taken at 540 nm (BioTek Synergy Hl microplat reade er). The aggregation of insulin leads to light scattering, which results in an increase in the measured absorbance. The time-to-aggregation (Za ) was defined as the timepoint when a 10% increase in transmittance from time zero was observed.

Circular dichroism: Circular dichroism was used to valida tethat aging wit h AC/DC excipient sdoes not result in changes to the secondary structure of insulin. Aged HUMULIN® (0.5, 1, 2, 4, and 6 months )or HUMULIN® aged wit h0.01 wt.% AC/DC excipient s(2, 4, and 6 months) were evaluated against an unaged HUMULIN® contro lor unaged HUMULIN® with 0.01 wt.% AC/DC excipient. Formulation samples were dilute d to 0.2 mg/mL in PBS (pH=7.4). Sample swere left to equilibrat fore 15 minutes at room temperature before measurement. Near-UV circular dichroism spectroscopy was performed at 20 °C wit ha J-815 CD Spectropolarimet er(Jasco Corporation) over a wavelengt rangeh of 200-260 nm using a 0.1 cm pathlength cell.

In vitro insuli ncellular activity assay: In vitro insuli nactivity was teste usingd the AKT phosphorylation pathway using AlphaLISA SureFire Ultra (Perkin-Elmer) kits for detection of phosphorylated AKT 1/2/3 (pS473) compared to total Aktl .HUMULIN®, Aged HUMULIN® (t = 6 months ),HUMULIN® + M0Ni23%, and Aged HUMULIN® + M0Ni23% (t = 6 months) formulations were tested. HUMULIN® + M0Ni23%, and Aged HUMULIN® + M0Ni23%(t = 6 months )formulations were tested. C2C12 mouse muscle myoblasts (ATCC CRL-1772) were cultured and were confirmed to be mycoplasma free prior to use. Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco; 4.5 g/L D-glucose , L-glutamine 110, mg/L sodium pyruvate) was supplemented with 10% feta lbovine serum (FBS) and 5% penicillin-streptomycin. Cells were grown in a 96-wel tissl ue culture plate for 24 hours (Seeding density= 25,000 cells/we inll 200 pL culture media). Prior to insulin stimulati on,the cells were washed twice wit h200 pL of unsupplemented DMEM and starved in 100 pL of unsupplemented DMEM overnight. The media was then removed and the cell swere stimulated wit h100 pL of insulin (i) HUMULIN®, (ii) Aged HUMULIN® (t = 6 months) , (iii) HUMULIN® + M0Ni23%, or (iv) Aged HUMULIN® + M0Ni23%(t = 6 months ),dilut edin unsupplemented DMEM, for 30 min while incubating at 37 °C. Cells were washed twice wit h100 pL of cold IX Tris-buffered saline before adding 100 pL of lysis buffer to each well and shaking for at least 10 minutes at room temperature to full y lyse cells. 30 pL of lysat wase transferred to a 96-wel whitel half-area plat fore each assay.

Assays were completed according to the manufacturer’s protocol. Plates were incubated at room temperatur ande read 18-20 hours after the additio nof the final assay reagents using a Tecan Infinite M1000 PRO plat ereader. Resul tswere plotted as a ratio of [pAKT]/[AKT] for each sampl e(n=3 cellular replicates) and an EC50 regression (log(agonist vs.) response (three parameters)) was plotte usingd GraphPad Prism 8.

Ethical approval of studi esincludin animg al experiments: All animal studi eswere performed in accordance wit hthe guidelines for the care and use of laboratory animals all; protocols (Protocol No. 32873) were approved by the Stanford Institutional Animal Care and Use Committee prior to the research being conducted.

Streptozotocin (STZ) induced model of diabetes in rats: Male Sprague Dawley rats (Charles River) were used for experiments. Animal studi eswere performed in accordance wit hthe guidelines for the care and use of laboratory animals ;all protocols were approved by the Stanford Institutional Animal Care and Use Committee The. protocol used for STZ inductio nadapted from the protocol by Wu and Huan, and have been previousl ydescribed. Male Sprague Dawley rats 180-250 g (8-10 weeks) were weighed and fasted the morning of treatment (6-8 hours) prior to treatment with STZ in the afternoon. Pre-weighed STZ was protected from light and dilut edto 10-20 mg/mL in 1 mL sodium citrat buffere (pH=4.5) immediately before injection. Rats were injected wit h STZ solution (65 mg/kg) intraperitoneall Ratsy. were given water containing 10% sucros e for 24 hours after administrati onof STZ. Three days after treatment wit hSTZ, rat blood glucose levels were tested for hyperglycemi avia tail vein blood collection using a handheld Bayer Contour Next glucose monitor (Bayer). Subsequen tglucose monitoring was performed daily. Diabetes was defined as having 3 consecutive blood glucos e measurements >300 mg/dL in non-fasted rats.

In vivo pharmacodynamics in diabetic rats: Diabetic rats were fasted for 4-6 hours.

For initial blood glucose studi esrats were injected subcutaneous (1.5ly U/kg) with the following formulations: (i) HUMULIN®, or (ii) HUMULIN® wit h0.01 wt.% AC/DC excipient (M0Ni23%, MpPhe8%, M0Phe6%). HUMULIN® formulations were tested at six aging time points of 0, 0.5, 1, 2, 4, and 6 months, and HUMULIN® wit hAC/DC excipient was tested at 0,2, 4, and 6 months of aging. The preparation of formulations was staggered so that all samples reached their endpoint age at the same time and all aging timepoint s coul dbe compared in the same cohort of rats. 32 rats wit hfasting glucose level >300s mg/dL were randomized to a formulati ongroup (8 rats/group and) each rat received that formulati onat all levels of aging (the order of the aging timepoints rats received was also randomized). For blood glucose studi esafter formulati onaging at 50 °C, rats were injected subcutaneous (1.5ly U/kg) wit hthe following formulations: (i) HUMULIN®, (ii) Aged HUMULIN® (t = 1 day), (iii) HUMULIN® + M0Ni23%, or (iv) Aged HUMULIN® + M0Ni23%(t = 4 days). 16 rats wit hfasting glucose level >300s mg/dL were randomized to either the HUMULIN® control group or the M0Ni23%group. Within both groups, the order that the aged formulations were given was also randomized and formulations were administered on separate experimental days. Before injection, baseline blood glucose was measured. After injection, blood was sampled every 30 minutes for 5 hours. Blood glucos e was measured using a handheld blood glucose monitor. The maximum change in blood glucose measured from baseline was used as a metric of bioactivity of each formulati onto assess in vivo bioactivity after aging.

In vivo pharmacokinetics in diabetic rats: Diabetic rats were fasted for 4-6 hours.

For pharmacokineti cstudi esrats were injected subcutaneous ly(1.5 U/kg) wit hthe following formulations: (i) HUMULIN®, (ii) Aged HUMULIN® (t = 6 months ),(iii) HUMULIN® + M0Ni23%, or (iv) Aged HUMULIN® + M0Ni23%(t = 6 months). 16 diabetic rats were randomized to a formulati ongroup: HUMULIN® or HUMULIN® + M0Ni23%(8 rats/group). Within each group, rats received both the fresh (t = 0 months) or aged (t = 6 months )formulations in a randomized order. After subcutaneous injection, blood was sampled every 15 minutes for 2 hours and blood was collected in serum tubes (Sarstedt) for analysis wit hELISA. Serum insulin concentrations were quantified using a Human Insuli nELISA kit (Mercodia).

Statisti cs:All data is shown as mean ± standar derror unless specified. For the in vitro activit assy ay (AKT) EC50 regression (log(agonist vs.) response (three parameters) ) was plotted using GraphPad Prism 8. GraphPad Prism 8 Extra sum-of-squares F-test was used to test if Log(EC50) differed between datasets. Data sets were compared in pairs, and Bonferroni post-hoc tests were used to adjus fort multiple comparisons (alpha=0.008). For blood glucose measurements a ,REML repeated measures mixed model was used to test for differences at different aging timepoints within a formulation (IMP Pro 14). Rat was included as a random effect and the age of the formulati onas a within-subje ctfixed effect.

A post-hoc Tukey HSD test was used on HUMULIN® formulations to determine statistical significance betwee naging timepoints.

Results Characterization of copolymers synthesized: The composition and molecula r weights of copolymer ssynthesized were determined via 1H NMR spectroscopy and SEC wit hpoly(ethylene glycol sta) ndards (Table 8).

Table 8. Characterization of Copolymers Carrier iW M,? Da wt.% wt.% by Dopant wt.% wt.% by (Target) NMR (Target) NMR (Da) (Da) (Exp) (Exp) MORPH 77 74.5b NIP 23 25.5b 3200 3800 1.19 MORPH 94 93.7C PHE 6 6.3C 2900 3400 1.17 MP AM 92 91d PHE 8 9d 5000 5400 1.08 3Determined using Size Exclusion Chromatography calibrated using polyethyle neglycol samples. b Weight percentages difficult to determine due to overlapping spectra. Weight percentages estimated from post-precipitated NMR spectr aby measuring the more resolved left half of the peak of Nipam (5= 4.0, 0.5 H), doubling it, and subtracti ngit from the unresolved peaks of MORPH and Nipam (5= 3.2-4.2, 7H (MORPH) 1H (Nipam)). 0Weight percentages calculated from post-precipitated NMR spectra of Morph (5= 3.3-3.7, 8H) and Phe (5= 7.6, 2H). dWeight percentages calculated from post-precipitated NMR spectr aof Mp (5= 3.1-3.5, 7H) and Phe (5= 7.6, 2H).

AC/DC excipient insulin stabilizing mechanism : Three amphiphilic AC/DC excipient sthat were shown to stabilize monomeric insulin were tested. These excipients were composed of either acryloylmorpholine (MORPH or Mo) or methoxypropylacrylam ide(MPAM) as a hydrophili ccarrier monomer copolymerize d wit h either N-Isopropylacrylami de(NIP or Ni) or phenylacrylamide (PHE) as a hydrophobic dopant monomer. To test whether these excipient spreferentially occupy the air-wate rinterface and consequentl inhiy bit insulin-insulin interactions occurring at these interfaces (FIG. 28), time-resolved surface tension and interfacia lrheology experiments were used with a model AC/DC excipient , poly(acryloylmorpholine77%-co-N- Isopropylacrylamide23%) (M0Ni23%), co-formulat edwit hcommercial HUMULIN® R (Eli Lilly) (FIG. 29).

Equilibrium surface tension measurements of HUMULIN® R, HUMULIN® R containing MoNi23%(0.01 wt.%), and a solution of M0Ni23% (0.01 wt.%) containing the same formulati onexcipient s(i.e., HUMULIN® R without the insulin) revealed that the presence of M0Ni23% resulted in surface tension values wel lbelow HUMULIN® R (approximately 42 vs. 47 mN/m, FIG. 29B). Moreover, a ten-fold increase in the M0Ni23% concentration (0.1 wt.% )further reduced the surface tension of the formulati on(FIG. 30).

The decrease in surface tension upon additio nof M0Ni23% to HUMULIN® indicates that there are more species at the interface when M0Ni23% and HUMULIN® are formulat ed togethe r,compared to HUMULIN® alone. The decreased surface tension concomitant wit h the increased concentration of M0Ni23%in the absence of HUMULIN® indicate sthat the surface is not saturated at 0.01 wt.% M0Ni23%. However, the surface tension is identica l for formulations of HUMULIN® and M0Ni23%and M0Ni23%with formulation excipients, indicating that there are similar number of molecula specir es at the interface regardles ofs the presence of HUMULIN®. Together , these surface tension experiment shelp demonstrat thate M0Ni23% preferentially adsorbs and dominates the air-water interface.

Interfacial shear rheology measurements demonstrat edthat addition of M0Ni23% (0.01 wt.%) to HUMULIN® R reduced interfacia lcomplex viscosity to below the detection limit of the instrument compared to HUMULIN® R, which exhibited values between 0.002 - 0.003 Pasm FIG. 29C). The complex viscosity of HUMULIN® is indicative of associative insulin-insulin interactions that can dissipate viscous energy at the interface.

While not quantitati ve,the lowerin gof the interfacial complex viscosity below instrument detection limits is indicative that the addition of M0Ni23% disrupts insulin-insulin interactions at the interface.

When this complex interface is subjected to interfacial stresse ands agitation, it is likel ythat these insulin-insuli assn ociations can nucleate amyloid fibril formation and lead to aggregation. Together ,the surface tension and interfacia lrheology experiment ssugges t a mechanism of AC/DC enhanced insulin stabilizatio whern e preferentia ladsorption of the AC/DC excipient to the air-wate rinterface disrupts insulin-insulin interactions.

AC/DC excipient sfor long-term stabilit ofy insulin: The capacity of the AC/DC excipient sM0Ni23%, poly(acryloylmorpholine94%-co-phenylacrylamide6%) (M0Phe6%) and poly(methoxypropylacrylamide92%-co-phenylacrylam ide8%)(MpPhe8%) to act as simple "drop-in" excipient sto stabilize HUMULIN® R through stressed aging was evaluated.

Formulations of HUMULIN® alone or HUMULIN® wit han AC/DC excipient added were prepared and aged for 0, 2, 4, or 6 months at 37 °C wit hconstant agitation (150 rpm on an orbital shaker plate) .The preparation of formulations was staggered so that all samples reached their endpoint age at the same time. Both visual inspection and a transmittance assays were used to determine if the insulin had aggregated (FIG. 31). Insuli naggregates scatt erlight, and thus aggregation can be defined as a change in transmittance greater than %. HUMULIN® alone began to aggregate after 2 weeks of stresse daging. In contrast , all insulin formulations containing AC/DC excipients M0Phe6%, MpPhe8%, and M0Ni23% at concentrations of 0.01, 0.1 or 0.5 wt.% did not show any signs of insulin aggregation over the course of the 6 month study, with the exception of MpPhe8% at 0.5 wt.% (FIG. 3 IB and FIG. 32). Thus, to minimize the amount of copolymer excipient in formulatio n, only the 0.01 wt.% formulations were used for the rest of the studi esreported here.

To furthe rvalidat thee transmittanc resulte s,which only assessed insuli n aggregation, in vitro activity was evaluated by assaying for phosphorylation of Ser473 on protein kinase B (AKT) after stimulatin C2C12g cell swit heither HUMULIN® or HUMULIN®® containing MoNi23%(0.01 wt.% )at both the 0 month and 6 month timepoints (FIGs. 31C-31D). Fresh formulations and the aged HUMULIN®+M0Ni23% formulati on showed equivalent bioactivity (HUMULIN® t=0 Log(EC50) = 2.252 ± 0.158; MoNi23%,t=0 Log(EC50) = 2.448 ± 0.186; M0Ni23%,t=6 Log(EC50) = 2.405 ± 0.158), whereas aged HUMULIN® R exhibited almost complet lose sof bioactivity (HUMULIN® t=6 Log(EC50) = 3.606 ± 0.139) (FIGs. 31C-31D).

While these in vitro AKT assay results supported the transmittance data ,insulin formulati onintegrit ywas further confirmed using circular dichroism to observe insulin secondary structure for each formulati ontimepoint (FIGs. 31E-31H). Formulations stabilize dwit hthe AC/DC excipient sexhibited no changes in secondary structure after stresse daging, whereas HUMULIN® alone had lost all structural features by 1 month.

These data corroborate both the transmittance and in vitro activit data.y Bioactivit ofy aged insulin in diabetic rats: To evaluate the integrit yof the aged insulin formulations in a functional setting in vivo, formulati onactivit iny diabetic rats was assessed. Administration of streptozotocin was used to induce insulin-dependent diabete sin a cohort of 32 mal erats. These rats were randomly assigned to one of four formulati ongroups :(i) HUMULIN®, or HUMULIN® comprising either (ii) M0Phe6%, (iii) MpPhe8%, or (iv) M0Ni23%at 0.01 wt.%, and each rat received that formulati onat each aging timepoint (0, 2, 4, 6 months). The preparation of formulations was staggered so that all samples reached their endpoint age at the same time and all aging timepoints could be compared in the same cohort of rats. Insuli nwas administere dsubcutaneous inly fasted rats (1.5U/kg) and blood glucose level swere measured every 30 minute s.Active formulations resulted in a distinc tinitial drop in blood glucose from extreme hyperglycemi athat reached a minimum in the range of normoglycemia between 60-100 minutes after administrati on(FIG. 33 and FIG. 34). After this phase, blood glucos leve els began to rise as insulin was cleared. In contrast formu, lations that appeared aggregated in the in vitro transmittance assays following aging did not show this distinc reduct tio nin glucos remie niscent of insulin action and instea dresulted in a gradual decrease in glucose level s.The gradua ldecrease in glucose may suggest that some of the insulin is initially trapped in reversible aggregates, and over time these aggregates dissociate and result in a slow-acti nginsulin effect. The maximum difference in blood glucose from baseline to the minimum glucose levels was plotted for each formulati onas a measure of formulati on potency. All copolymer-stabilized formulations showed no difference in activity between aging timepoints but, HUMULIN® alone demonstrated a large difference betwee naging timepoints (F3,21=23.83, P<0.0001), where a post-hoc Tukey HSD test revealed that HUMULIN® after 2, 4, and 6 months of aging had decreased activit comparedy to fresh HUMULIN® (t=0 months). These observations were corroborated by evaluati onof insulin pharmacokinetics where, no differences were observed between fresh HUMULIN® R (t=0 months )and HUMULIN®+M0Ni23% initiall (t=y 0 months) and after 6 months of aging, but a decrease in exposure was observed for the aged HUMULIN® (t=6 months) (FIG. 33F and FIG. 35). These data suggest that AC/DC excipient sfunction as stabilizing ingredients for commercial formulations such as HUMULIN® R without altering the insuli n pharmacokinetic sor pharmacodynamics.

High temperature aging of insulin formulations: To determine the capacity of AC/DC excipient sto improve insulin cold-chain resilience, the extent of stabilit imbuedy by M0Ni23%under extreme manufacturing and distribut ionconditions (37 °C and 50 °C wit hconstant agitation) was evaluated (FIG. 36). Temperatures were selected to represent the temperatur one a hot summer day (37 °C), and the upper temperature range that a shipping container or truck without refrigeration or insulati oncoul dreach during the peak of summer (50 °C). HUMULIN® R can be purchased in 10mL glass vial sthat are packaged and shipped in cardboard boxes (FIG. 36 A and FIG. 37). M0Ni23% (0.01 wt.%) was added to new vial sof HUMULIN® R using a syringe (dilutio fromn 100 U/mL to 95U/mL to allow addition of copolymer; control vial was diluted wit hwater) and the vial swere then replaced in the original cardboard packaging wit hthe package insert (FIG. 37A). The cardboard packaging was affixed to a rotary shaker inside a temperature-controlled incubator and agitated at 150 RPM (FIG. 37B).

Visual inspectio ncombined wit ha transmittanc asse ay were used as the primary measures of insulin integrity (FIG. 36A). These assays were consistent with our earlier experiment sthat demonstrat edthat the transmittance readings correlat ewel lwit hboth in vitro and in vivo functional activity assays. At 37 °C, HUMULIN® alone began to show visual changes in opacity at day 1 and became fully opaque by day 2. In contrast when, formulat edwit hM0Ni23%, the insulin formulation showed no visual changes in opacity until day 56 and remained below a 10% change in transmittance for 56 days. At 50 °C, commercial HUMULIN® became fully opaque within one day. In contrast formul, ati on wit hM0Ni23% extende dstabilit undery these extreme conditions to past 4 days before the formulati onbecame cloudy on day 5. These qualitat iveobservations were consistent wit h quantitati transve mittanc reade ings (FIGs. 36B-36C).

To verify functional insulin activity after aging at 50 °C in vivo, these formulations were evaluated in diabetic rats. The ability of (i) HUMULIN® (t = 0 day), (ii) aged HUMULINE (t = 1 day), (iii) HUMULIN® wit hM0Ni23% (t = 0 day), or (iv) aged HUMULIN® with M0Ni23%(t = 4 days) to decrease glucose levels was measured in fasted diabetic rats. After subcutaneous administrati onof formulations (1.5U/kg), blood glucose levels were measured every 30 minutes (FIG. 36D). HUMULIN® (t = 0 day), HUMULIN® wit hM0Ni23% (t = 0 day), and aged HUMULIN® wit hM0Ni23% (t = 4 day) demonstrat ed an initial blood glucose drop that reached a minimum between 60-100 minutes after injection. These resul tswere consistent wit hactive formulations in earlier experiments.

This characteristi glucosc edrop was absent in rats who received aged HUMULIN® (t = 1 day), consistent wit hinactive formulations in earlier experiments. The maximum difference in glucos efrom baseline was also plotte ford each formulati onas a metric of formulati onpotency (FIG. 36E).

Statistica anall ysis identified a difference between the potency of these formulations (F3,18.18=10.71, P=0.0003), whereby a post-hoc Tukey HSD test reveale d that aged HUMULIN® alone had significantly decreased activit ycompared to the other formulations. In contrast there, was no statistical difference betwee nunaged HUMULIN®, unaged HUMULIN® wit hM0Ni23%, as well as aged HUMULIN® wit hM0Ni23% after stresse agingd at 50 °C for 4 days.

Various modifications of the invention ,in additio ton those described herein, wil l be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

Claims (69)

CLAIMED IS:

1. A polyacrylamide-based copolymer comprising: a water-soluble carrier monomer selected from the group consisting of A-(3- methoxypropoyl)acrylamide (MPAM), 4-acryloylmorpholine (MORPH), N,N- 5 dimethylacrylamide (DMA), A-hydroxy ethyl acrylamide (HEAM), and acrylamide (AM); and a functional dopant monomer selected from the group consisting of N- [tris(hydroxymethyl)-methyl]acrylamide (TRI), 2-acrylamido-2-methylpropane sulfonic acid (AMP), (3-acrylamidopropyl)trimethylammonium chloride (TMA), N- 10 isopropyl acrylamide (NIP), A-Zc/7-butylacrylamide (TEA), and N-phenyl acrylamide (PHE).

2. The copolymer of claim 1, wherein: the water-soluble carrier monomer is selected from the group consisting of A-(3- methoxypropoyl)acrylamide (MPAM) and 4-acryloylmorpholine (MORPH); and 15 the functional dopant monomer is selected from the group consisting of N- isopropyl acrylamide (NIP) and A-phenyl acrylamide (PHE).

3. The copolymer of claim 1 or 2, wherein the weight percent (wt%) of the water-soluble carrier monomer is about 70% to about 98%, about 75% to about 95%, or about 80% to about 95%. 20

4. The copolymer of any one of claims 1-3, wherein the weight percent (wt%) of the functional dopant monomer is about 2% to about 30%, about 5% to about 25%, or about 5% to about 20%.

5. The copolymer of claim 1 or 2, wherein the functional dopant monomer is NIP and is about 5% to about 30%, about 10% to about 28%, about 2% to about 30%, 25 about 5% to about 26%, or about 20% to about 26% by weight of the copolymer.

6. The copolymer of claim 5, wherein the water-soluble carrier monomer is MORPH.

7. The copolymer of claim 5, wherein the water-soluble carrier monomer is MP AM. WO 2021/211976 PCT/US2021/027693 -115-

8. The copolymer of claim 1, wherein the functional dopant monomer is selected from the group consisting of AMP, TMA, TBA, and PHE and is about 2% to about 16%, about 12% to about 15%, about 5% to about 15%, or about 6% to about 14% by weight of the copolymer. 5 9. The copolymer of claim 8, wherein the water-soluble carrier monomer is

9.MORPH.

10. The copolymer of claim 8, wherein the water-soluble carrier monomer is MP AM

11. The copolymer of any one of claims 8-10, wherein the functional dopant 10 monomer is PHE.

12. The copolymer of claim 1, wherein the functional dopant monomer is TRI and is about 3% to about 17%, about 4% to about 6%, about 7% to about 12%, or about 13% to about 17% by weight of the copolymer.

13. The copolymer of any one of claims 1-12, wherein the degree of 15 polymerization is about 10 to about 500, about 20 to about 200, or about 50.

14. The copolymer of any one of claims 1-13, wherein the molecular weight of the copolymer is about 1,000 to about 40,000 g/mol, about 1,000 to about 30,000 g/mol, about 2,000 to about 10,000 g/mol, about 3,000 to about 7,000 g/mol, or about 4,000 to about 6,000 g/mol. 20

15. The copolymer of any one of claims 1-14, wherein the functional dopant monomer is hydrophobic.

16. The copolymer of any one of claims 1-15, wherein the copolymer is amphiphilic.

17. A polyacrylamide-based copolymer comprising: 25 a water-soluble carrier monomer comprising an acrylamide reactive moiety; and a functional dopant monomer comprising an acrylamide reactive moiety; wherein: WO 2021/211976 PCT/US2021/027693 -116- the weight percent (wt%) of the water-soluble carrier monomer is about 70% to about 98%; the weight percent (wt%) of the functional dopant monomer is about 2% to about 30%; 5 the average molecular weight (Mn) of the polyacrylamide-based copolymer is about 1,000 g/mol to about 30,000 g/mol; and the degree of polymerization is about 10 to about 250.

18. The copolymer of claim 17, wherein the water-soluble carrier monomer is selected from the group consisting of 7V-(3-methoxypropoyl)acrylamide (MPAM), 4- 10 acryloylmorpholine (MORPH), 7V,7V-dimethylacrylamide (DMA), 7V-hydroxyethyl acrylamide (HEAM), and acrylamide (AM).

19. The copolymer of claim 18, wherein the water-soluble carrier monomer is selected from the group consisting of 7V-(3-methoxypropoyl)acrylamide (MPAM) and 4- acryloylmorpholine (MORPH). 15

20. The copolymer of any one of claims 17-19, wherein the functional dopant monomer is selected from the group consisting of 7V-[tris(hydroxymethyl)- methyl]acrylamide (TRI), 2-acrylamido-2-methylpropane sulfonic acid (AMP), (3- acrylamidopropyl)trimethylammonium chloride (TMA), 7V-isopropylacrylamide (NIP), TV-te/7-butylacrylamide (TEA), and 7V-phenyl acrylamide (PHE).

21. The copolymer of claim 20, wherein the functional dopant monomer is selected from the group consisting of 7V-isopropylacrylamide (NIP) and N- phenyl acrylamide (PHE).

22. A composition comprising the polyacrylamide-based copolymer of any one of claims 1-21 and a pharmaceutically acceptable excipient. 25

23. A composition comprising the copolymer of any one of claims 1-21, wherein the composition is a cosmetic product, a hair product, a lotion, a food product, a veterinary product, or a nutritional product.

24. A composition comprising the copolymer of any one of claims 1-21 and a protein. WO 2021/211976 PCT/US2021/027693 -117-

25. The composition of claim 24, wherein the protein is a protein susceptible to aggregation in an aqueous medium.

26. The composition of claim 24 or 25, wherein the protein is selected from the group consisting of antibodies and fragments thereof, cytokines, chemokines, 5 hormones, vaccine antigens, cancer antigens, adjuvants, and combinations thereof.

27. The composition of any one of claims 24-26, wherein the composition comprises the protein in a concentration at least two times greater, at least three times greater, at least four times greater, or at least five times greater than the concentration of the same protein in the composition without the copolymer. 10

28. The composition of claim 26 or 27, wherein the protein is a monoclonal antibody.

29. The composition of any one of claims 24-28, wherein the protein is insulin, or an analog thereof.

30. The composition of claim 29, wherein the insulin, or an analog thereof, is 15 selected from the group consisting of porcine insulin, bovine insulin, feline insulin, human insulin, recombinant insulin, insulin lispro, HUMALOG®, insulin glargine, LANTUS®, insulin detemir, LEVEMIR®, ACTRAPID®, modern insulin, NOVORAPID®, VELOSULIN®, HUMULIN® M3, HYPURIN®, INSUMAN®, INSULATARD®, MIXTARD® 30, MIXTARD® 40, MIXTARD® 50, insulin aspart, 20 insulin glulisine, insulin isophane, insulin degludec, insulin icodec, insulin zinc extended, NO VOLIN® R, HUMULIN® R, HUMULIN® R regular U-500, NO VOLIN® N, HUMULIN® N, RELION®, AFREZZA®, HUMULIN® 70/30, NO VOLIN® 70/30, NOVOLOG® 70/30, HUMULIN® 50/50, HUMALOG® mix 75/25, insulin aspart protamine-insulin aspart, insulin lispro protamine-insulin lispro, insulin lispro protamine- 25 insulin lispro, human insulin NPH-human insulin regular, insulin degludec-insulin aspart, and combinations thereof.

31. The composition of claim 30, wherein the insulin, or an analog thereof, is a human insulin or a recombinant human insulin. WO 2021/211976 PCT/US2021/027693 -118-

32. The composition of any one of claims 29-31, wherein the insulin, or an analog thereof, comprises about 50% or greater, about 60% or greater, about 70% or greater, about 80% or greater, about 90% or greater, or about 99% or greater insulin present in monomeric form. 5 33. The composition of any one of claims 29-32, wherein the insulin concentration is about 0.34 mg/mL (10 U/mL) to about 34 mg/mL (1000 U/mL), about

33.7 mg/mL (50 U/mL) to about 17 mg/mL (500 U/mL), about 3.4 mg/mL (100 U/mL), about 6.8 mg/mL (200 U/mL), or about 10.2 mg/mL (300 U/mL).

34. The composition of any one of claims 24-33, wherein the copolymer 10 concentration is about 0.0001% to about 5%, about 0.001% to about 1% by weight, about 0.005% to about 0.5% by weight, about 0.005% to about 0.02% by weight, about

35.01% to about 0.2% by weight, about 0.1% to about 0.4% by weight, or about 0.2% to about 0.3% by weight, about 0.005% by weight, about 0.01% by weight, about 0.05% by weight, about 0.1% by weight, or about 1% by weight of the composition. 15 35. The composition of any one of claims 24-34, wherein the composition comprises one or more of an aqueous buffer, tonicity modifier, and preservative, and combinations thereof.

36. The composition of any one of claims 24-35, wherein the pH of the composition is about 4 to about 9 or about 7.4. 20

37. The composition of any one of claims 29-36, further comprising glucagon, a GLP-1 agonist, a glucose-dependent insulinotropic polypeptide (GIP), or a dual GIP and GLP-1 agonist.

38. The composition of claim 37, wherein the GLP-1 agonist is selected from the group consisting of lixisenatide, liraglutide, albiglutide, dulaglutide, exenatide, 25 extended-release exenatide, and semaglutide.

39. The composition of claim 37, wherein the dual GIP and GLP-1 agonist is tirzepatide. WO 2021/211976 PCT/US2021/027693 -119-

40. The composition of any one of claims 29-36, further comprising amylin, or an analog thereof.

41. The composition of claim 40, wherein the amylin analog is pramlintide.

42. The composition of claim 40 or 41, wherein the amylin, or an analog 5 thereof, and insulin, or an analog thereof are present in a ratio of about 1:1 to about 1:20, about 1:1 to about 1:15, about 1:1 to about 1:10, about 1:1 to about 1:6, or about 1:20, about 1:15, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, or about 1:1.

43. The composition of any one of claims 24-42, suitable for human or 10 veterinary administration.

44. A composition comprising: about 0.01 wt% of a polyacrylamide-based copolymer comprising: about 74% to about 80% by weight of a MORPH carrier monomer; and about 20% to about 26% by weight of a NIP dopant monomer; and 15 about 100 U/mL insulin, or an analog thereof.

45. A composition comprising: about 0.01 wt% of a polyacrylamide-based copolymer comprising: about 74% to about 80% by weight of a MORPH carrier monomer; and about 20% to about 26% by weight of a NIP dopant monomer; 20 about 100 U/mL insulin, or an analog thereof; and about 0.5 mg/mL to about 0.6 mg/mL pramlintide.

46. The composition of claim 45, wherein the pH of the composition is about 6 to about 8.

47. The composition of claim 45 or 46, wherein the insulin, or analog thereof, 25 is substantially present in monomeric form.

48. The composition of any one of claims 29-47, wherein the copolymer stabilizes the insulin such that the insulin exhibits increased stability when stored at room WO 2021/211976 PCT/US2021/027693 -120- temperature as compared to the same insulin composition that does not contain the copolymer.

49. The composition of claim 48, wherein the increased stability is at least 10- fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at 5 least 40-fold, at least 45-fold, at least 50-fold, or greater as compared to the same insulin composition that does not contain the copolymer.

50. The composition of any one of claims 29-49, wherein the insulin, or an analog thereof, comprises about 60% or greater, about 70% or greater, about 80% or greater, about 90% or greater, or about 99% or greater insulin in monomeric form. 10

51. The composition of any one of claims 22-50, wherein the composition is aqueous.

52. A method of treating an elevated glucose level in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition of any one of claims 29-51, wherein the elevated glucose level is associated 15 with insulin deficiency in the subject.

53. A method of managing the blood glucose level in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition of any one of claims 29-51.

54. A method for increasing thermal stability of a protein formulation, 20 comprising adding about 0.0005 wt% to about 5 wt% of the copolymer of any one of claims 1-21 to the protein formulation.

55. A method for increasing stability of a protein formulation, comprising adding about 0.005 wt% to about 5 wt% of the copolymer of any one of claims 1-21 to the protein formulation. 25

56. A method for reducing the rate of aggregation of a protein in an aqueous composition, comprising adding about 0.005 wt% to about 5 wt% of the copolymer of any one of claims 1-21 to the protein formulation. WO 2021/211976 PCT/US2021/027693 -121-

57. The method of any one of claims 54-56, wherein the protein is a protein that tends to aggregate in an aqueous medium.

58. The method of any one of claims 54-57, wherein the protein is selected from the group consisting of antibodies and fragments thereof, cytokines, chemokines, 5 hormones, vaccine antigens, cancer antigens, adjuvants and combinations thereof.

59. The method of any one of claims 54-58, wherein the protein is a monoclonal antibody.

60. The method of any one of claims 54-58, wherein the protein is a vaccine antigen. 10

61. The method of any one of claims 54-58, wherein the protein is insulin, or an analog thereof.

62. The method of claim 61, wherein the insulin, or an analog thereof, is selected from the group consisting of porcine insulin, bovine insulin, feline insulin, human insulin, recombinant insulin, insulin lispro, HUMALOG®, insulin glargine, LANTUS®, insulin detemir, LEVEMIR®, ACTRAPID", modern insulin, 15 NOVORAPID®, VELOSULIN®, HUMULIN® M3, HYPURIN®, INSUMAN®, INSULATARD®, MIXTARD® 30, MIXTARD® 40, MIXTARD® 50, insulin aspart, insulin glulisine, insulin isophane, insulin degludec, insulin icodec, insulin zinc extended, NO VOLIN® R, HUMULIN® R, HUMULIN® R regular U-500, NO VOLIN® 20 N, HUMULIN® N, RELION®, AFREZZA®, HUMULIN® 70/30, NO VOLIN® 70/30, NOVOLOG® 70/30, HUMULIN® 50/50, HUMALOG® mix 75/25, insulin aspart protamine-insulin aspart, insulin lispro protamine-insulin lispro, insulin lispro protamine- insulin lispro, human insulin NPH-human insulin regular, insulin degludec-insulin aspart, and combinations thereof. 25

63. The method of claim 62, wherein the insulin, or an analog thereof, is a human insulin or a recombinant human insulin.

64. The method of any one of claims 61-63, wherein the insulin is human insulin. WO 2021/211976 PCT/US2021/027693 -122-

65. The method of claim 64, wherein the copolymer increases the time to aggregation of the human insulin formulation by at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, or at least 50-fold when stored at 37 °C as assessed by a transmittance assay as compared 5 to a human insulin formulation that does not contain the copolymer.

66. The method of claim 64 or 65, wherein the copolymer increases the time to aggregation of the human insulin formulation by at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, or at least 50-fold when stored at 50 °C as assessed by a transmittance assay as 10 compared to a human insulin formulation that does not contain the copolymer.

67. The method of any one of claims 64 to 66 , wherein addition of the copolymer maintains the in vitro bioactivity of the human insulin formulation for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, or at least 6 months. 15

68. The method of any one of claims 52-67, wherein the composition is aqueous.

69. The method of any one of claims 52-68, wherein the composition is administered via infusion pumps or artificial pancreas closed-loop systems.